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Sample records for aquaporin isoforms involved

  1. Differential water permeability and regulation of three aquaporin 4 isoforms

    DEFF Research Database (Denmark)

    Fenton, Robert A.; Moeller, Hanne B; Zelenina, Marina

    2010-01-01

    Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the b...

  2. New isoforms of rat Aquaporin-4

    DEFF Research Database (Denmark)

    Moe, Svein Erik; Sorbo, Jan Gunnar; Søgaard, Rikke;

    2008-01-01

    an intracellular localization when expressed in cell lines and do not transport water when expressed in Xenopus oocytes. In contrast, the largest of the new isoforms, AQP4e, which contains a novel N-terminal domain, is localized at the plasma membrane in cell lines and functions as a water transporter in Xenopus...

  3. Short-term control of maize cell and root water permeability through plasma membrane aquaporin isoforms.

    Science.gov (United States)

    Hachez, Charles; Veselov, Dmitry; Ye, Qing; Reinhardt, Hagen; Knipfer, Thorsten; Fricke, Wieland; Chaumont, François

    2012-01-01

    Although it is widely accepted that aquaporins are involved in the regulation of root water uptake, the role of specific isoforms in this process is poorly understood. The mRNA expression and protein level of specific plasma membrane intrinsic proteins (PIPs) were analysed in Zea mays in relation to cell and root hydraulic conductivity. Plants were analysed during the day/night period, under different growth conditions (aeroponics/hydroponics) and in response to short-term osmotic stress applied through polyethylene glycol (PEG). Higher protein levels of ZmPIP1;2, ZmPIP2;1/2;2, ZmPIP2;5 and ZmPIP2;6 during the day coincided with a higher water permeability of root cortex cells during the day compared with night period. Similarly, plants which were grown under aeroponic conditions and which developed a hypodermis ('exodermis') with Casparian bands, effectively forcing more water along a membranous uptake path across roots, showed increased levels of ZmPIP2;5 and ZmPIP1;2 in the rhizodermis and exodermis. When PEG was added to the root medium (2-8 h), expression of PIPs and cell water permeability in roots increased. These data support a role of specific PIP isoforms, in particular ZmPIP1;2 and ZmPIP2;5, in regulating root water uptake and cortex cell hydraulic conductivity in maize.

  4. Aquaporin-4 Mz Isoform: Brain Expression, Supramolecular Assembly and Neuromyelitis Optica Antibody Binding%Aquaporin-4Mz Isoform: Brain Expression, Supramolecular Assembly and Neuromyelitis Optica Antibody Binding

    Institute of Scientific and Technical Information of China (English)

    Andrea Rossi; Jonathan M.Crane; A.S.Verkman

    2011-01-01

    水通道蛋白4(AQP4)表达于大脑和脊髓的星形胶质细胞.2种主要的AQP4亚型,Ml和M23,均有表达,具有不同的翻译起始点.研究表明,一种较新的亚型Mz表达于大鼠,其翻译起始点位于M1的翻译起始点上游126bp.通过C端标记的抗 AQP4 抗体SDS和非变性胶免疫印迹技术,大鼠大脑中的Mz被检测到为39 kDa大小的条带.Mz因其提前终止密码子,在人类和小鼠大脑中并不表达.通过单粒子追踪和非变性胶电泳技术检测,发现大鼠Mz可形成正交粒子阵列(OAPs).本文发现,Mz与M1类似,在细胞浆膜内迅速扩散,并不形成OAPs.但是,当与M23共表达时,Mz通过与M23形成异四聚体可与OAPs关联.意外的是,Mz表达的细胞极弱地与视神经脊髓炎自身抗体(NMO-IgG)结合,小于M1表达细胞的5倍.切割分析提示,Met-1上游的31-41残基参与NMO-IgG与Mz之间较弱的结合.总之,Mz AQP4在大鼠中低量表达,但不表达于人和小鼠的大脑;自身无法形成OAPs,除非与M23 AQP4形成异四聚体;因AQP4/NMO-IgG结合点的N端功能,一般不能与NMO-IgG结合.%Water channel aquaporin-4(AQP4) is expressed in astrocytes throughout brain and spinal cord. Two major AQP4 isoforms are expressed, Ml and M23, having different translation initiation sites. A longer isoform (Mz)has been reported in rat with translation initiation 126-bp upstream from that of M1. By immunoblot analysis of SDS and native gels probed with a C-terminus anti-AQP4 antibody, Mz was detected in rat brain as a distinct band of size -39 kDa. Mz was absent in human and mouse brain because of in-frame stop codons. The ability of rat Mz to form orthogonal arrays of particles (OAPs) was investigated by single particle tracking and native gel electrophoresis. We found that Mz, like M1, diffused rapidly in the cell plasma membrane and did not form OAPs. However, when co-ex-pressed with M23, Mz associated in OAPs by forming heterotetramers with M23. Unexpectedly, Mz

  5. Involvement of aquaporin channels in water extrusion from biosilica during maturation of sponge siliceous spicules.

    Science.gov (United States)

    Wang, Xiaohong; Müller, Werner E G

    2015-08-01

    Aquaporins are a family of small, pore-forming, integral cell membrane proteins. This ancient protein family functions as water channels and is found in all kingdoms (including archaea, eubacteria, fungi, plants, and animals). We discovered that in sponges aquaporin plays a novel role during the maturation of spicules, their skeletal elements. Spicules are synthesized enzymatically via silicatein following a polycondensation reaction. During this process, a 1:1 stoichiometric release of water per one Si-O-Si bond formed is produced. The product of silicatein, biosilica, is a fluffy, soft material that must be hardened in order to function as a solid rod. Using the model of the demosponge species Suberites domuncula Olivi, 1792, which expresses aquaporin, cDNA was cloned and the protein was heterologously expressed. The sponge aquaporin is grouped with the type 8 aquaporins. The function of the sponge aquaporin can be blocked by Mn-sulfate (MnSO4) and mercury chloride (HgCl2). Microscopic and functional studies suggest that aquaporin is involved in removal of the reaction water at the site where siliceous spicules are formed. Another molecule that is likely to be involved in biosilica maturation is the mucin/nidogen-like polypeptide. cDNA has also been cloned from S. domuncula. Experimental studies suggest that water extrusion/suctioning from biosilica after enzymatic synthesis during spicule formation involves both aquaporin-mediated water channeling and "polymerization-induced phase separation" facilitated by the mucin/nidogen-like polypeptide.

  6. Patterns of antibody binding to aquaporin-4 isoforms in neuromyelitis optica.

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    Simone Mader

    Full Text Available BACKGROUND: Neuromyelitis optica (NMO, a severe demyelinating disease, represents itself with optic neuritis and longitudinally extensive transverse myelitis. Serum NMO-IgG autoantibodies (Abs, a specific finding in NMO patients, target the water channel protein aquaporin-4 (AQP4, which is expressed as a long (M-1 or a short (M-23 isoform. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to analyze serum samples from patients with NMO and controls for the presence and epitope specificity of IgG and IgM anti-AQP4 Abs using an immunofluorescence assay with HEK293 cells expressing M-1 or M-23 human AQP4. We included 56 patients with definite NMO (n = 30 and high risk NMO (n = 26, 101 patients with multiple sclerosis, 27 patients with clinically isolated syndromes (CIS, 30 patients with systemic lupus erythematosus (SLE or Sjögren's syndrome, 29 patients with other neurological diseases and 47 healthy controls. Serum anti-AQP4 M-23 IgG Abs were specifically detected in 29 NMO patients, 17 patients with high risk NMO and two patients with myelitis due to demyelination (CIS and SLE. In contrast, IgM anti-AQP4 Abs were not only found in some NMO and high risk patients, but also in controls. The sensitivity of the M-23 AQP4 IgG assay was 97% for NMO and 65% for high risk NMO, with a specificity of 100% compared to the controls. Sensitivity with M-1 AQP4 transfected cells was lower for NMO (70% and high risk NMO (39%. The conformational epitopes of M-23 AQP4 are the primary targets of NMO-IgG Abs, whereas M-1 AQP4 Abs are developed with increasing disease duration and number of relapses. CONCLUSIONS: Our results confirm M-23 AQP4-IgG Abs as reliable biomarkers in patients with NMO and high risk syndromes. M-1 and M-23 AQP4-IgG Abs are significantly associated with a higher number of relapses and longer disease duration.

  7. Aquaporin 4 is a Ubiquitously Expressed Isoform in the Dogfish (Squalus acanthias) Shark

    Science.gov (United States)

    Cutler, Christopher P; MacIver, Bryce; Cramb, Gordon; Zeidel, Mark

    2012-01-01

    The dogfish ortholog of aquaporin 4 (AQP4) was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5′ and 3′ RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3%) of homology with higher vertebrate sequences but lower levels of homology to Agnathan (38.2%) or teleost (57.5%) fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ). Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver > gill >  intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW) or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney, or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressing-oocytes, exhibited significantly increased osmotic water permeability (Pf) compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing-oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species. PMID:22291652

  8. Aquaporin 4 is a ubiquitously expressed isoform in the dogfish (Squalus acanthias shark.

    Directory of Open Access Journals (Sweden)

    Christopher P Cutler

    2012-01-01

    Full Text Available The dogfish orthologue of aquaporin 4 (AQP4 was amplified from cDNA using degenerate PCR followed by cloning and sequencing. The complete coding region was then obtained using 5’ and 3’ RACE techniques. Alignment of the sequence with AQP4 amino acid sequences from other species showed that dogfish AQP4 has high levels (up to 65.3% of homology with higher vertebrate sequences but lower levels of homology to agnathan (38.2% or teleost (57.5% fish sequences. Northern blotting indicated that the dogfish mRNA was approximately 3.2 kb and was highly expressed in the rectal gland (a shark fluid secretory organ. Semi-quantitative PCR further indicates that AQP4 is ubiquitous, being expressed in all tissues measured but at low levels in certain tissues, where the level in liver>gill> intestine. Manipulation of the external environmental salinity of groups of dogfish showed that when fish were acclimated in stages to 120% seawater (SW or 75% SW, there was no change in AQP4 mRNA expression in either rectal gland, kidney or esophagus/cardiac stomach. Whereas quantitative PCR experiments using the RNA samples from the same experiment, showed a significant 63.1% lower abundance of gill AQP4 mRNA expression in 120% SW-acclimated dogfish. The function of dogfish AQP4 was also determined by measuring the effect of the AQP4 expression in Xenopus laevis oocytes. Dogfish AQP4 expressingoocytes, exhibited significantly increased osmotic water permeability (Pf compared to controls, and this was invariant with pH. Permeability was not significantly reduced by treatment of oocytes with mercury chloride, as is also the case with AQP4 in other species. Similarly AQP4 expressing oocytes did not exhibit enhanced urea or glycerol permeability, which is also consistent with the water-selective property of AQP4 in other species.

  9. Genetic deletion of laminin isoforms β2 and γ3 induces a reduction in Kir4.1 and aquaporin-4 expression and function in the retina.

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    Petra G Hirrlinger

    Full Text Available Glial cells such as retinal Müller glial cells are involved in potassium ion and water homeostasis of the neural tissue. In these cells, inwardly rectifying potassium (Kir channels and aquaporin-4 water channels play an important role in the process of spatial potassium buffering and water drainage. Moreover, Kir4.1 channels are involved in the maintenance of the negative Müller cell membrane potential. The subcellular distribution of Kir4.1 and aquaporin-4 channels appears to be maintained by interactions with extracellular and intracellular molecules. Laminins in the extracellular matrix, dystroglycan in the membrane, and dystrophins in the cytomatrix form a complex mediating the polarized expression of Kir4.1 and aquaporin-4 in Müller cells.The aim of the present study was to test the function of the β2 and γ3 containing laminins in murine Müller cells. We used knockout mice with genetic deletion of both β2 and γ3 laminin genes to assay the effects on Kir4.1 and aquaporin-4. We studied protein and mRNA expression by immunohistochemistry, Western Blot, and quantitative RT-PCR, respectively, and membrane currents of isolated cells by patch-clamp experiments. We found a down-regulation of mRNA and protein of Kir4.1 as well as of aquaporin-4 protein in laminin knockout mice. Moreover, Müller cells from laminin β2 and γ3 knockout mice had reduced Kir-mediated inward currents and their membrane potentials were more positive than those in age-matched wild-type mice.These findings demonstrate a strong impact of laminin β2 and γ3 subunits on the expression and function of both aquaporin-4 and Kir4.1, two important membrane proteins in Müller cells.

  10. A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers.

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    Bienert, Gerd P; Cavez, Damien; Besserer, Arnaud; Berny, Marie C; Gilis, Dimitri; Rooman, Marianne; Chaumont, François

    2012-07-01

    AQPs (aquaporins) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Among the different plant AQPs, PIPs (plasma membrane intrinsic proteins), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. In the present study, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulfide bond between two monomers. Although this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of ZmPIP1s (Zm is Zea mays), ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.

  11. Involvement of water channel Aquaporin 5 in H2S-induced pulmonary edema.

    Science.gov (United States)

    Xu, Chunyang; Jiang, Lei; Zou, Yuxia; Xing, Jingjing; Sun, Hao; Zhu, Baoli; Zhang, Hengdong; Wang, Jun; Zhang, Jinsong

    2017-01-01

    Acute exposure to hydrogen sulfide (H2S) poses a significant threat to life, and the lung is one of the primary target organs of H2S. However, the mechanisms involved in H2S-induced acute pulmonary edema are poorly understood. This study aims to investigate the effects of H2S on the expression of water channel aquaporin 5 (AQP5) and to elucidate the signaling pathways involved in AQP5 regulation. In an in vivo study, C57BL6 mice were exposed to sub-lethal concentrations of inhaled H2S, and histological injury of the lungs and ultrastructure injury of the epithelial cells were evaluated. With real-time PCR and western blot assays, we found that H2S exposure contributed to a significant decrease in AQP5 expression both in murine lung tissue and the A549 cell line, and the ERK1/2 and p38 MAPK signaling pathways were demonstrated to be implicated in AQP5 regulation. Therefore, adjusting AQP5 protein levels could be considered a therapeutic strategy for the treatment of APE induced by H2S and other hazardous gases.

  12. Aquaporin JcPIP2 is Involved in Drought Responses in Jatropha curcas

    Institute of Scientific and Technical Information of China (English)

    Ying ZHANG; Yunxiao WANG; Luding JIANG; Ying XU; Yingchun WANG; Daihua LU; Fang CHEN

    2007-01-01

    Water channel proteins, aquaporins, play fundamental roles in transmembrane water movements in plants. A new full-length cDNA encoding aquaporin was isolated from the seedlings of Jatropha curcas.The gene of the plasma membrane intrinsic protein (PIP) from J. curcas (JcPIP2) contained an 843 bp open reading frame encoding a protein of 280 amino acids. The amino acid sequence showed 94% identity with Ricinus communis PIP. Injection of JcPIP2 complementary RNA into Xenopus oocytes increased 10-fold the osmotic water permeability of the oocytes. Immunodetection of JcPIP2 with anti-JcPIP2 antibody indicated that this protein is ubiquitously located in all tested tissues of the plant. To investigate the relationship between aquaporins and drought resistance in J. curcas, the abundance of JcPIP2 was examined in seedlings of two J. curcas populations, Gao You CSC63 and YanBian S1, under water deficit with PEG6000. Under field conditions, those two populations, Gao You CSC63 was resistant to water deficit, but YanBian S1 was sensitive to water deprivation. With the increasing degree of drought stress, JcPIP2 level increased in seedlings of Gao You CSC63, whereas there was no significant change in seedlings of YanBian S1. Compared with YanBian S1, GaoYou CSC63 also showed higher root hydraulic conductivity and lower decreasing trend in the seedlings under water deficit. These results indicated that JcPIP2 probably played a role in drought resistance in J. curcas.

  13. Aquaporin JcPIP2 is involved in drought responses in Jatropha curcas.

    Science.gov (United States)

    Zhang, Ying; Wang, Yunxiao; Jiang, Luding; Xu, Ying; Wang, Yingchun; Lu, Daihua; Chen, Fang

    2007-10-01

    Water channel proteins, aquaporins, play fundamental roles in transmembrane water movements in plants. A new full-length cDNA encoding aquaporin was isolated from the seedlings of Jatropha curcas. The gene of the plasma membrane intrinsic protein (PIP) from J. curcas (JcPIP2) contained an 843 bp open reading frame encoding a protein of 280 amino acids. The amino acid sequence showed 94% identity with Ricinus communis PIP. Injection of JcPIP2 complementary RNA into Xenopus oocytes increased 10-fold the osmotic water permeability of the oocytes. Immunodetection of JcPIP2 with anti-JcPIP2 antibody indicated that this protein is ubiquitously located in all tested tissues of the plant. To investigate the relationship between aquaporins and drought resistance in J. curcas, the abundance of JcPIP2 was examined in seedlings of two J. curcas populations, GaoYou CSC63 and YanBian S1, under water deficit with PEG6000. Under field conditions, those two populations, GaoYou CSC63 was resistant to water deficit, but YanBian S1 was sensitive to water deprivation. With the increasing degree of drought stress, JcPIP2 level increased in seedlings of GaoYou CSC63, whereas there was no significant change in seedlings of YanBian S1. Compared with YanBian S1, GaoYou CSC63 also showed higher root hydraulic conductivity and lower decreasing trend in the seedlings under water deficit. These results indicated that JcPIP2 probably played a role in drought resistance in J. curcas.

  14. Functional involvement of VAMP/synaptobrevin-2 in cAMP-stimulated aquaporin 2 translocation in renal collecting duct cells.

    Science.gov (United States)

    Gouraud, Sabine; Laera, Antonia; Calamita, Giuseppe; Carmosino, Monica; Procino, Giuseppe; Rossetto, Ornella; Mannucci, Roberta; Rosenthal, Walter; Svelto, Maria; Valenti, Giovanna

    2002-09-15

    The involvement of soluble N-ethylmaleimide sensitive factor-attachment protein receptor (SNARE) proteins in the cAMP-induced exocytosis of aquaporin 2 (AQP2)-containing vesicles was investigated in AQP2-transfected renal CD8 cells. RT-PCR and western blot analysis confirmed the presence of the SNARE homologs VAMP/synaptobrevin-2, syntaxin-1, syntaxin-4 and SNAP-23 in CD8 cells. Tetanus neurotoxin (TeNT) was efficient in cleaving synaptobrevin-like protein both in vitro and in intact CD8 cells incubated with the toxin. TeNT treatment in intact CD8 cells completely abolished cAMP-stimulated AQP2 targeting to the plasma membrane, as assessed by quantification of cell-surface immunoreactivity to an anti-AQP2 antibody raised against a peptide reproducing the extracellular AQP2 C-loop. These results represent the first evidence for the functional involvement of VAMP-2 in cAMP-induced AQP2 exocytosis in renal cells.

  15. Involvement of aquaporin-3 in epidermal growth factor receptor signaling via hydrogen peroxide transport in cancer cells.

    Science.gov (United States)

    Hara-Chikuma, Mariko; Watanabe, Sachiko; Satooka, Hiroki

    2016-03-18

    Aquaporin 3 (AQP3), a water/glycerol channel protein, is capable of transporting hydrogen peroxide (H2O2). Here, we show that AQP3-mediated intracellular H2O2 is involved in epidermal growth factor (EGF)-induced cell signaling and its dependent cell function in the EGF receptor (EGFR)-positive cancer cell lines A431 and H1666. AQP3 knockdown suppressed the transport into the cells of extracellular H2O2 produced in response to EGF in A431 and H1666 cells. EGF-induced Erk and Akt activation, which occurred through SHP2 and/or PTEN modulation, was impaired by AQP3 knockdown. Cell growth and migration induced by EGF stimulation were attenuated in AQP3 knockdown cells compared with those in control cells. Coincidentally, tumor growth of A431 cell xenografts in immunodeficient mice was decreased by AQP3 knockdown. Accordingly, a xenograft with AQP3 knockdown A431 cells significantly enhanced the survival of recipient mice compared with the transplantation with control cells. In addition, AQP3 associated with EGFR and NADPH oxidase 2, which we propose is linked to AQP3 producing a localized increase in intracellular H2O2 to function as a second messenger during EGFR cell signaling. Therefore, our findings suggest that AQP3 is required for EGF-EGFR cell signaling in cancer cells and is a therapeutic target for cancer progression.

  16. Aquaporins in complex tissues

    DEFF Research Database (Denmark)

    Hamann, S; Zeuthen, T; La Cour, M

    1998-01-01

    Multiple physiological fluid movements are involved in vision. Here we define the cellular and subcellular sites of aquaporin (AQP) water transport proteins in human and rat eyes by immunoblotting, high-resolution immunocytochemistry, and immunoelectron microscopy. AQP3 is abundant in bulbar conj......, predicting specific roles for each in the complex network through which water movements occur in the eye....

  17. MzPIP2;1: An Aquaporin Involved in Radial Water Movement in Both Water Uptake and Transportation, Altered the Drought and Salt Tolerance of Transgenic Arabidopsis.

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    Lin Wang

    Full Text Available Plants are unavoidably subjected to various abiotic stressors, including high salinity, drought and low temperature, which results in water deficit and even death. Water uptake and transportation play a critical role in response to these stresses. Many aquaporin proteins, localized at different tissues, function in various transmembrane water movements. We targeted at the key aquaporin in charge of both water uptake in roots and radial water transportation from vascular tissues through the whole plant.The MzPIP2;1 gene encoding a plasma membrane intrinsic protein was cloned from salt-tolerant apple rootstock Malus zumi Mats. The GUS gene was driven by MzPIP2;1 promoter in transgenic Arabidopsis. It indicated that MzPIP2;1 might function in the epidermal and vascular cells of roots, parenchyma cells around vessels through the stems and vascular tissues of leaves. The ectopically expressed MzPIP2;1 conferred the transgenic Arabidopsis plants enhanced tolerance to slight salt and drought stresses, but sensitive to moderate salt stress, which was indicated by root length, lateral root number, fresh weight and K+/Na+ ratio. In addition, the possible key cis-elements in response to salt, drought and cold stresses were isolated by the promoter deletion experiment.The MzPIP2;1 protein, as a PIP2 aquaporins subgroup member, involved in radial water movement, controls water absorption and usage efficiency and alters transgenic plants drought and salt tolerance.

  18. Mutual interactions between aquaporins and membrane components

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    MCarmen Martinez-Ballesta

    2016-08-01

    Full Text Available During the last years, a number of studies have been focused on the structural evaluation of protein complexes in order to get mechanistic insights into how proteins communicate at the molecular level within the cell. Specific sites of protein-aquaporin interaction have been evaluated and new regulations of aquaporins described based on these associations. Aquaporin isoforms heterotetramerizations are considered as novel regulatory mechanisms for plasma membrane (PIPs and tonoplast (TIPs proteins, influencing their intrinsic permeability and trafficking dynamics in the adaptive response to changing environmental conditions. However, protein-protein interaction is an extensive theme that is difficult to tackle and new methodologies of physical interactions are being used in approaches to its study. Bimolecular fluorescence complementation (BiFC and the identification of cross-linked peptides based on tandem mass spectra, which resulted complementary to other methodologies such as heterologous expression, co-precipitation assays or confocal fluorescence microscopy, have been discussed in this review. The chemical composition or physical characteristics of the lipid bilayer also influences many aspects of membrane aquaporins, including their functionality. The molecular driving forces stabilizing the observed lipid positions around aquaporins could define their activity, which could alter the conformational properties. Therefore, an integrative approach to the relevance of the membrane-aquaporin interaction to different processes related to plant cell physiology is shown. Finally, the interactions between aquaporins and copolymer matrixes or biological compounds offer an opportunity for the functional incorporation of aquaporins into new biotechnological advances.

  19. New insights into the regulation of aquaporins by the arbuscular mycorrhizal symbiosis in maize plants under drought stress and possible implications for plant performance.

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    Bárzana, Gloria; Aroca, Ricardo; Bienert, Gerd Patrick; Chaumont, François; Ruiz-Lozano, Juan Manuel

    2014-04-01

    The relationship between modulation by arbuscular mycorrhizae (AM) of aquaporin expression in the host plant and changes in root hydraulic conductance, plant water status, and performance under stressful conditions is not well known. This investigation aimed to elucidate how the AM symbiosis modulates the expression of the whole set of aquaporin genes in maize plants under different growing and drought stress conditions, as well as to characterize some of these aquaporins in order to shed further light on the molecules that may be involved in the mycorrhizal responses to drought. The AM symbiosis regulated a wide number of aquaporins in the host plant, comprising members of the different aquaporin subfamilies. The regulation of these genes depends on the watering conditions and the severity of the drought stress imposed. Some of these aquaporins can transport water and also other molecules which are of physiological importance for plant performance. AM plants grew and developed better than non-AM plants under the different conditions assayed. Thus, for the first time, this study relates the well-known better performance of AM plants under drought stress to not only the water movement in their tissues but also the mobilization of N compounds, glycerol, signaling molecules, or metalloids with a role in abiotic stress tolerance. Future studies should elucidate the specific function of each aquaporin isoform regulated by the AM symbiosis in order to shed further light on how the symbiosis alters the plant fitness under stressful conditions.

  20. Involvement of yeast HSP90 isoforms in response to stress and cell death induced by acetic acid.

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    Silva, Alexandra; Sampaio-Marques, Belém; Fernandes, Angela; Carreto, Laura; Rodrigues, Fernando; Holcik, Martin; Santos, Manuel A S; Ludovico, Paula

    2013-01-01

    Acetic acid-induced apoptosis in yeast is accompanied by an impairment of the general protein synthesis machinery, yet paradoxically also by the up-regulation of the two isoforms of the heat shock protein 90 (HSP90) chaperone family, Hsc82p and Hsp82p. Herein, we show that impairment of cap-dependent translation initiation induced by acetic acid is caused by the phosphorylation and inactivation of eIF2α by Gcn2p kinase. A microarray analysis of polysome-associated mRNAs engaged in translation in acetic acid challenged cells further revealed that HSP90 mRNAs are over-represented in this polysome fraction suggesting preferential translation of HSP90 upon acetic acid treatment. The relevance of HSP90 isoform translation during programmed cell death (PCD) was unveiled using genetic and pharmacological abrogation of HSP90, which suggests opposing roles for HSP90 isoforms in cell survival and death. Hsc82p appears to promote survival and its deletion leads to necrotic cell death, while Hsp82p is a pro-death molecule involved in acetic acid-induced apoptosis. Therefore, HSP90 isoforms have distinct roles in the control of cell fate during PCD and their selective translation regulates cellular response to acetic acid stress.

  1. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism.

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    González-Mariscal, Isabel; Krzysik-Walker, Susan M; Doyle, Máire E; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D; Witek, Rafal P; O'Connell, Jennifer F; Egan, Josephine M

    2016-09-19

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism.

  2. Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism

    Science.gov (United States)

    González-Mariscal, Isabel; Krzysik-Walker, Susan M.; Doyle, Máire E.; Liu, Qing-Rong; Cimbro, Raffaello; Santa-Cruz Calvo, Sara; Ghosh, Soumita; Cieśla, Łukasz; Moaddel, Ruin; Carlson, Olga D.; Witek, Rafal P.; O’Connell, Jennifer F.; Egan, Josephine M.

    2016-01-01

    Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism. PMID:27641999

  3. Identification of the human UDP-glucuronosyltransferase isoforms involved in the glucuronidation of the phytochemical ferulic acid.

    Science.gov (United States)

    Li, Xiaojun; Shang, Liang; Wu, Yaohua; Abbas, Suzanne; Li, Dong; Netter, Patrick; Ouzzine, Mohamed; Wang, Hui; Magdalou, Jacques

    2011-01-01

    Ferulic acid (FA), a member of the hydroxycinnamate family, is an abundant dietary antioxidant that may offer beneficial effects against cancer, cardiovascular disease, diabetes, osteoarthritis and Alzheimer's disease. In this study, evidence for sulfation and glucuronidation of FA was investigated upon incubation with human liver microsomes and cytosol. Two main glucuronides, M1 (ether O-glucuronide) and M2 (ester acylglucuronide), were formed with a similar affinity (apparent K(m) 3.53 and 5.15 mM, respectively). A phenol sulfoconjugate was also formed with a higher affinity (K(m) 0.53 mM). Identification of the UDP-glucuronosyltransferase (UGT) isoforms involved in FA glucuronidation was investigated with 12 human recombinant enzymes. FA was mainly glucuronidated by UGT1A isoforms and by UGT2B7. UGT1A4, 2B4, 2B15 and 2B17 failed to glucuronidate the substance. Examination of the kinetic constants revealed that FA was mainly glucuronidated by UGT1A1 at the two nucleophilic groups. UGT1A3 was able to glucuronidate these two positions with the same, but low, efficiency. UGT1A6 and 1A8 were involved in the formation of the ether glucuronide only, whereas UGT1A7, 1A10 and 2B7 preferentially glucuronidated the carboxyl group. Moreover, octyl gallate, a marker substrate of UGT1A1, competitively inhibited FA glucuronidation mediated by this isoform. Altogether, the results suggest that FA glucuronidation is primarily mediated by UGT1A1.

  4. Indoxyl Sulfate as a Mediator Involved in Dysregulation of Pulmonary Aquaporin-5 in Acute Lung Injury Caused by Acute Kidney Injury

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    Nozomi Yabuuchi

    2016-12-01

    Full Text Available High mortality of acute kidney injury (AKI is associated with acute lung injury (ALI, which is a typical complication of AKI. Although it is suggested that dysregulation of lung salt and water channels following AKI plays a pivotal role in ALI, the mechanism of its dysregulation has not been elucidated. Here, we examined the involvement of a typical oxidative stress-inducing uremic toxin, indoxyl sulfate (IS, in the dysregulation of the pulmonary predominant water channel, aquaporin 5 (AQP-5, in bilateral nephrectomy (BNx-induced AKI model rats. BNx evoked AKI with the increases in serum creatinine (SCr, blood urea nitrogen (BUN and serum IS levels and exhibited thickening of interstitial tissue in the lung. Administration of AST-120, clinically-used oral spherical adsorptive carbon beads, resulted in a significant decrease in serum IS level and thickening of interstitial tissue, which was accompanied with the decreases in IS accumulation in various tissues, especially lung. Interestingly, a significant decrease in AQP-5 expression of lung was observed in BNx rats. Moreover, the BNx-induced decrease in pulmonary AQP-5 protein expression was markedly restored by oral administration of AST-120. These results suggest that BNx-induced AKI causes dysregulation of pulmonary AQP-5 expression, in which IS could play a toxico-physiological role as a mediator involved in renopulmonary crosstalk.

  5. Protection of Vascular Endothelial Growth Factor to Brain Edema Following Intracerebral Hemorrhage and Its Involved Mechanisms: Effect of Aquaporin-4.

    Directory of Open Access Journals (Sweden)

    Heling Chu

    Full Text Available Vascular endothelial growth factor (VEGF has protective effects on many neurological diseases. However, whether VEGF acts on brain edema following intracerebral hemorrhage (ICH is largely unknown. Our previous study has shown aquaporin-4 (AQP4 plays an important role in brain edema elimination following ICH. Meanwhile, there is close relationship between VEGF and AQP4. In this study, we aimed to test effects of VEGF on brain edema following ICH and examine whether they were AQP4 dependent. Recombinant human VEGF165 (rhVEGF165 was injected intracerebroventricularly 1 d after ICH induced by microinjecting autologous whole blood into striatum. We detected perihemotomal AQP4 protein expression, then examined the effects of rhVEGF165 on perihemotomal brain edema at 1 d, 3 d, and 7 d after injection in wild type (AQP4(+/+ and AQP4 knock-out (AQP4(-/- mice. Furthermore, we assessed the possible signal transduction pathways activated by VEGF to regulate AQP4 expression via astrocyte cultures. We found perihemotomal AQP4 protein expression was highly increased by rhVEGF165. RhVEGF165 alleviated perihemotomal brain edema in AQP4(+/+ mice at each time point, but had no effect on AQP4(-/- mice. Perihemotomal EB extravasation was increased by rhVEGF165 in AQP4(-/- mice, but not AQP4(+/+ mice. RhVEGF165 reduced neurological deficits and increased Nissl's staining cells surrounding hemotoma in both types of mice and these effects were related to AQP4. RhVEGF165 up-regulated phospharylation of C-Jun amino-terminal kinase (p-JNK and extracellular signal-regulated kinase (p-ERK and AQP4 protein in cultured astrocytes. The latter was inhibited by JNK and ERK inhibitors. In conclusion, VEGF reduces neurological deficits, brain edema, and neuronal death surrounding hemotoma but has no influence on BBB permeability. These effects are closely related to AQP4 up-regulation, possibly through activating JNK and ERK pathways. The current study may present new insights to

  6. Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: Involvement of NFAT5

    Science.gov (United States)

    Vogler, Stefanie; Reichenbach, Andreas; Wiedemann, Peter; Bringmann, Andreas; Kohen, Leon

    2015-01-01

    Purpose High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration. Neovascular retinal diseases are associated with edema. Various factors and channels, including vascular endothelial growth factor (VEGF) and aquaporins (AQPs), influence neovascularization and the development of edema. Therefore, we determined whether extracellular hyperosmolarity alters the expression of VEGF and AQPs in cultured human retinal pigment epithelial (RPE) cells. Methods Human RPE cells obtained within 48 h of donor death were prepared and cultured. Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Alterations in gene expression and protein secretion were determined with real-time RT–PCR and ELISA, respectively. The levels of signaling proteins and nuclear factor of activated T cell 5 (NFAT5) were determined by western blotting. DNA binding of NFAT5 was determined with EMSA. NFAT5 was knocked down with siRNA. Results Extracellular hyperosmolarity stimulated VEGF gene transcription and the secretion of VEGF protein. Hyperosmolarity also increased the gene expression of AQP5 and AQP8, induced the phosphorylation of p38 MAPK and ERK1/2, increased the expression of HIF-1α and NFAT5, and induced the DNA binding of NFAT5. The hyperosmotic expression of VEGF was dependent on the activation of p38 MAPK, ERK1/2, JNK, PI3K, HIF-1, and NFAT5. The hyperosmotic induction of AQP5 was in part dependent on the activation of p38 MAPK, ERK1/2, NF-κB, and NFAT5. Triamcinolone acetonide inhibited the hyperosmotic expression of VEGF but not AQP5. The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia. Conclusions Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells. The data suggest that high salt intake resulting in osmotic stress may aggravate neovascular retinal diseases and

  7. Aquaporins in Salivary Glands: From Basic Research to Clinical Applications

    Directory of Open Access Journals (Sweden)

    Christine Delporte

    2016-01-01

    Full Text Available Salivary glands are involved in saliva secretion that ensures proper oral health. Aquaporins are expressed in salivary glands and play a major role in saliva secretion. This review will provide an overview of the salivary gland morphology and physiology of saliva secretion, and focus on the expression, subcellular localization and role of aquaporins under physiological and pathophysiological conditions, as well as clinical applications involving aquaporins. This review is highlighting expression and localization of aquaporins in human, rat and mouse, the most studied species and is pointing out possible difference between major salivary glands, i.e., parotid, submandibular and sublingual glands.

  8. Involvement of mitogen-activated protein kinase pathways in expression of the water channel protein aquaporin-4 after ischemia in rat cortical astrocytes.

    Science.gov (United States)

    Nito, Chikako; Kamada, Hiroshi; Endo, Hidenori; Narasimhan, Purnima; Lee, Yong-Sun; Chan, Pak H

    2012-09-20

    Brain edema after ischemic brain injury is a key determinant of morbidity and mortality. Aquaporin-4 (AQP4) plays an important role in water transport in the central nervous system and is highly expressed in brain astrocytes. However, the AQP4 regulatory mechanisms are poorly understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs), which are involved in changes in osmolality, might mediate AQP4 expression in models of rat cortical astrocytes after ischemia. Increased levels of AQP4 in primary cultured astrocytes subjected to oxygen-glucose deprivation (OGD) and 2 h of reoxygenation were observed, after which they immediately decreased at 0 h of reoxygenation. Astrocytes exposed to OGD injury had significantly increased phosphorylation of three kinds of MAPKs. Treatment with SB203580, a selective p38 MAPK inhibitor, or SP600125, a selective c-Jun N-terminal kinase inhibitor, significantly attenuated the return of AQP4 to its normal level, and SB203580, but not SP600125, significantly decreased cell death. In an in vivo study, AQP4 expression was upregulated 1-3 days after reperfusion, which was consistent with the time course of p38 phosphorylation and activation, and decreased by the p38 inhibition after transient middle cerebral artery occlusion (MCAO). These results suggest that p38 MAPK may regulate AQP4 expression in cortical astrocytes after ischemic injury.

  9. Expression Analysis of Sugarcane Aquaporin Genes under Water Deficit

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    Manassés Daniel da Silva

    2013-01-01

    Full Text Available The present work is a pioneer study specifically addressing the aquaporin transcripts in sugarcane transcriptomes. Representatives of the four aquaporin subfamilies (PIP, TIP, SIP, and NIP, already described for higher plants, were identified. Forty-two distinct aquaporin isoforms were expressed in four HT-SuperSAGE libraries from sugarcane roots of drought-tolerant and -sensitive genotypes, respectively. At least 10 different potential aquaporin isoform targets and their respective unitags were considered to be promising for future studies and especially for the development of molecular markers for plant breeding. From those 10 isoforms, four (SoPIP2-4, SoPIP2-6, OsPIP2-4, and SsPIP1-1 showed distinct responses towards drought, with divergent expressions between the bulks from tolerant and sensitive genotypes, when they were compared under normal and stress conditions. Two targets (SsPIP1-1 and SoPIP1-3/PIP1-4 were selected for validation via RT-qPCR and their expression patterns as detected by HT-SuperSAGE were confirmed. The employed validation strategy revealed that different genotypes share the same tolerant or sensitive phenotype, respectively, but may use different routes for stress acclimation, indicating the aquaporin transcription in sugarcane to be potentially genotype-specific.

  10. Intestinal epithelial restitution. Involvement of specific laminin isoforms and integrin laminin receptors in wound closure of a transformed model epithelium

    DEFF Research Database (Denmark)

    Lotz, M M; Nusrat, A; Madara, J L;

    1997-01-01

    Disruptions in the mucosal lining of the gastrointestinal tract reseal by epithelial cell migration, a process termed restitution. We examined the involvement of laminin isoforms and their integrin receptors in restitution using the intestinal epithelial cell line T84. T84 cells express primarily...... laminins 5, 6, and 7 as indicated by immunostaining using laminin subunit-specific monoclonal antibodies (MAbs). A MAb (BM2) specific for the laminin alpha 3 subunit, a component of laminins 5, 6, and 7, completely inhibited the closure of mechanical wounds in T84 monolayers. Confocal microscopy using MAbs...... BM2 (laminin alpha 3 subunit) and 6F12 (laminin beta 3 subunit) revealed that laminin-5 is deposited in a basal matrix that extends into the wound. The MAbs 4E10 (laminin beta 1 subunit) and C4 (laminin beta 2 subunit) stained the lateral membranes between T84 cells. This staining was enhanced...

  11. Significance of oxygen transport through aquaporins

    Science.gov (United States)

    Zwiazek, Janusz J.; Xu, Hao; Tan, Xiangfeng; Navarro-Ródenas, Alfonso; Morte, Asunción

    2017-01-01

    Aquaporins are membrane integral proteins responsible for the transmembrane transport of water and other small neutral molecules. Despite their well-acknowledged importance in water transport, their significance in gas transport processes remains unclear. Growing evidence points to the involvement of plant aquaporins in CO2 delivery for photosynthesis. The role of these channel proteins in the transport of O2 and other gases may also be more important than previously envisioned. In this study, we examined O2 permeability of various human, plant, and fungal aquaporins by co-expressing heterologous aquaporin and myoglobin in yeast. Two of the most promising O2-transporters (Homo sapiens AQP1 and Nicotiana tabacum PIP1;3) were confirmed to facilitate O2 transport in the spectrophotometric assay using yeast protoplasts. The over-expression of NtPIP1;3 in yeasts significantly increased their O2 uptake rates in suspension culture. In N. tabacum roots subjected to hypoxic hydroponic conditions, the transcript levels of the O2-transporting aquaporin NtPIP1;3 significantly increased after the seven-day hypoxia treatment, which was accompanied by the increase of ATP levels in the apical root segments. Our results suggest that the functional significance of aquaporin-mediated O2 transport and the possibility of controlling the rate of transmembrane O2 transport should be further explored. PMID:28079178

  12. The aquaporin family of water channel proteins in clinical medicine.

    Science.gov (United States)

    Lee, M D; King, L S; Agre, P

    1997-05-01

    The aquaporins are a family of membrane channel proteins that serve as selective pores through which water crosses the plasma membranes of many human tissues and cell types. The sites where aquaporins are expressed implicate these proteins in renal water reabsorption, cerebrospinal fluid secretion and reabsorption, generation of pulmonary secretions, aqueous humor secretion and reabsorption, lacrimation, and multiple other physiologic processes. Determination of the aquaporin gene sequences and their chromosomal locations has provided insight into the structure and pathophysiologic roles of these proteins, and primary and secondary involvement of aquaporins is becoming apparent in diverse clinical disorders. Aquaporin-1 (AQP1) is expressed in multiple tissues including red blood cells, and the Colton blood group antigens represent a polymorphism on the AQP1 protein. AQP2 is restricted to renal collecting ducts and has been linked to congenital nephrogenic diabetes insipidus in humans and to lithium-induced nephrogenic diabetes insipidus and fluid retention from congestive heart failure in rat models. Congenital cataracts result from mutations in the mouse gene encoding the lens homolog Aqp0 (Mip). The present understanding of aquaporin physiology is still incomplete; identification of additional members of the aquaporin family will affect future studies of multiple disorders of water distribution throughout the body. In some tissues, the aquaporins may participate in the transepithelial movement of fluid without being rate limiting, so aquaporins may be involved in clinical disorders without being causative. As outlined in this review, our challenge is to identify disease states in which aquaporins are involved, to define the aquaporins' roles mechanistically, and to search for ways to exploit this information therapeutically.

  13. Two p90 ribosomal S6 kinase isoforms are involved in the regulation of mitotic and meiotic arrest in Artemia.

    Science.gov (United States)

    Duan, Ru-Bing; Zhang, Li; Chen, Dian-Fu; Yang, Fan; Yang, Jin-Shu; Yang, Wei-Jun

    2014-06-06

    There are multiple isoforms of p90 ribosomal S6 kinase (RSK), which regulate diverse cellular functions such as cell growth, proliferation, maturation, and motility. However, the relationship between the structures and functions of RSK isoforms remains undetermined. Artemia is a useful model in which to study cell cycle arrest because these animals undergo prolonged diapauses, a state of obligate dormancy. A novel RSK isoform was identified in Artemia, which was termed Ar-Rsk2. This isoform was compared with an RSK isoform that we previously identified in Artemia, termed Ar-Rsk1. Ar-Rsk2 has an ERK-docking motif, whereas Ar-Rsk1 does not. Western blot analysis revealed that Ar-Rsk1 was activated by phosphorylation, which blocked meiosis in oocytes. Knockdown of Ar-Rsk1 reduced the level of phosphorylated cdc2 and thereby suppressed cytostatic factor activity. This indicates that Ar-Rsk1 regulates the cytostatic factor in meiosis. Expression of Ar-Rsk2 was down-regulated in Artemia cysts in which mitosis was arrested. Knockdown of Ar-Rsk2 resulted in decreased levels of cyclin D3 and phosphorylated histone H3, and the production of pseudo-diapause cysts. This indicates that Ar-Rsk2 regulates mitotic arrest. PLK and ERK RNAi showed that Ar-Rsk2, but not Ar-Rsk1, could be activated by PLK-ERK in Artemia. This is the first study to report that RSK isoforms with and without an ERK-docking motif regulate mitosis and meiosis, respectively. This study provides insight into the relationship between the structures and functions of RSK isoforms.

  14. Tim50a, a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis

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    Robinson Melvin L

    2005-07-01

    Full Text Available Abstract Background The Cajal body (CB is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs, which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation. Results In this report, we identify a minor isoform of the mitochondrial Tim50, Tim50a, as a coilin interacting protein. The Tim50a transcript can be detected in some cancer cell lines and normal brain tissue. The Tim50a protein differs only from Tim50 in that it contains an additional 103 aa N-terminal to the translation start of Tim50. Importantly, a putative nuclear localization signal is found within these 103 residues. In contrast to Tim50, which localizes to the cytoplasm and mitochondria, Tim50a is strictly nuclear and is enriched in speckles with snRNPs. In addition to coilin, Tim50a interacts with snRNPs and SMN. Competition binding experiments demonstrate that coilin competes with Sm proteins of snRNPs and SMN for binding sites on Tim50a. Conclusion Tim50a may play a role in snRNP biogenesis given its cellular localization and protein interaction characteristics. We hypothesize that Tim50a takes part in the release of snRNPs and SMN from the CB.

  15. Expression of Fragaria vesca PIP Aquaporins in Response to Drought Stress: PIP Down-Regulation Correlates with the Decline in Substrate Moisture Content

    OpenAIRE

    Nada Šurbanovski; Sargent, Daniel J.; Else, Mark A.; Simpson, David W.; Hanma Zhang; Grant, Olga M.

    2013-01-01

    PIP aquaporin responses to drought stress can vary considerably depending on the isoform, tissue, species or level of stress; however, a general down-regulation of these genes is thought to help reduce water loss and prevent backflow of water to the drying soil. It has been suggested therefore, that it may be necessary for the plant to limit aquaporin production during drought stress, but it is unknown whether aquaporin down-regulation is gradual or triggered by a particular intensity of the ...

  16. XBAT35, a Novel Arabidopsis RING E3 Ligase Exhibiting Dual Targeting of Its Splice Isoforms,Is Involved in Ethylene-Mediated Regulation of Apical Hook Curvature

    Institute of Scientific and Technical Information of China (English)

    Sofia D.Carvalho; Rita Saraiva; Teresa M.Maia; Isabel A.Abreu; Paula Duque

    2012-01-01

    The Arabidopsis XBAT35 is one of five structurally related ankyrin repeat-containing Really interesting New Gene (RING) E3 ligases involved in ubiquitin-mediated protein degradation,which plays key roles in a wide range of cellular processes.Here,we show that the XBAT35 gene undergoes alternative splicing,generating two transcripts that are constitutively expressed in all plant tissues.The two splice variants derive from an exon skipping event that excludes an in-frame segment from the XBAT35 precursor mRNA,giving rise to two protein isoforms that differ solely in the presence of a nuclear localization signal (NLS).Transient expression assays indicate that the isoform lacking the NLS localizes in the cytoplasm of plant cells,whereas the other is targeted to the nucleus,accumulating in nuclear speckles.Both isoforms are functional E3 ligases,as assessed by in vitro ubiquitination assays.Two insertion mutant alleles and RNA-interference (RNAi) silencing lines for XBAT35 display no evident phenotypes under normal growth conditions,but exhibit hypersensitivity to the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) during apical hook exaggeration in the dark,which is rescued by an inhibitor of ethylene perception.Independent expression of each XBAT35 splice variant in the mutant background indicates that the two isoforms may differentially contribute to apical hook formation but are both functional in this ethylene-mediated response.Thus,XBAT35 defines a novel player in ethylene signaling involved in negatively regulating apical hook curvature,with alternative splicing controlling dual targeting of this E3 ubiquitin ligase to the nuclear and cytoplasmic compartments.

  17. The phosphoarginine energy-buffering system of trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations.

    Directory of Open Access Journals (Sweden)

    Frank Voncken

    Full Text Available Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3. Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide 'SNL'. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90% of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed.

  18. Crystal structures of barley thioredoxin h isoforms HvTrxh1 and HvTrxh2 reveal features involved in protein recognition and possibly in discriminating the isoform specificity

    DEFF Research Database (Denmark)

    Maeda, Kenji; Hägglund, Per; Finnie, Christine;

    2008-01-01

    H-type thioredoxins (Trxs) constitute a particularly large Trx sub-group in higher plants. Here, the crystal structures are determined for the two barley Trx h isoforms, HvTrxh1 and HvTrxh2, in the partially radiation-reduced state to resolutions of 1.7 angstrom, and for HvTrxh2 in the oxidized...... state to 2.0 angstrom. The two Trxs have a sequence identity of 51% and highly similar fold and active-site architecture. Interestingly, the four independent molecules in the crystals of HvTrxh1 form two relatively large and essentially identical protein-protein interfaces. In each interface, a loop......) of the bound loop segment in the proximity of the Cys40 thiol. The interaction involves three characteristic backbone-backbone hydrogen bonds in an antiparallel beta-sheet-like arrangement, similar to the arrangement observed in the structure of an engineered, covalently bound complex between Trx...

  19. The peripheral binding of 14-3-3γ to membranes involves isoform-specific histidine residues.

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    Helene J Bustad

    Full Text Available Mammalian 14-3-3 protein scaffolds include seven conserved isoforms that bind numerous phosphorylated protein partners and regulate many cellular processes. Some 14-3-3-isoforms, notably γ, have elevated affinity for membranes, which might contribute to modulate the subcellular localization of the partners and substantiate the importance of investigating molecular mechanisms of membrane interaction. By applying surface plasmon resonance we here show that the binding to phospholipid bilayers is stimulated when 14-3-3γ is complexed with its partner, a peptide corresponding to the Ser19-phosphorylated N-terminal region of tyrosine hydroxylase. Moreover, membrane interaction is dependent on salts of kosmotropic ions, which also stabilize 14-3-3γ. Electrostatic analysis of available crystal structures of γ and of the non-membrane-binding ζ-isoform, complemented with molecular dynamics simulations, indicate that the electrostatic potential distribution of phosphopeptide-bound 14-3-3γ is optimal for interaction with the membrane through amphipathic helices at the N-terminal dimerization region. In addition, His158, and especially His195, both specific to 14-3-3γ and located at the convex lateral side, appeared to be pivotal for the ligand induced membrane interaction, as corroborated by site-directed mutagenesis. The participation of these histidine residues might be associated to their increased protonation upon membrane binding. Overall, these results reveal membrane-targeting motifs and give insights on mechanisms that furnish the 14-3-3γ scaffold with the capacity for tuned shuffling from soluble to membrane-bound states.

  20. Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene

    Institute of Scientific and Technical Information of China (English)

    Guang-Jing Hu; Jia Chen; Xiao-Nan Zhao; Jia-Jia Xu; Dong-Qing Guo; Ming Lu; Ming Zhu

    2013-01-01

    Trans-splicing,a process involving the cleavage and joining of two separate transcripts,can expand the transcriptome and proteome in eukaryotes.Chimeric RNAs generated by trans-splicing are increasingly described in literatures.The widespread presence of antibiotic resistance genes in natural environments and human intestines is becoming an important challenge for public health.Certain antibiotic resistance genes,such as ampicillin resistance gene (Amp),are frequently used in recombinant plasmids.Until now,trans-splicing involving recombinant plasmid-derived exogenous transcripts and endogenous cellular RNAs has not been reported.Acyl-CoA:cholesterol acyltransferase 1 (ACAT1) is a key enzyme involved in cellular cholesterol homeostasis.The 4.3-kb human ACAT1 chimeric mRNA can produce 50-kDa and 56-kDa isoforms with different enzymatic activities.Here,we show that human ACAT1 56-kDa isoform is produced from an mRNA species generated through the trans-splicing of an exogenous transcript encoded by the antisense strand of Ampr (asAmp) present in common Ampr-plasmids and the 4.3-kb endogenous ACAT1 chimeric mRNA,which is presumably processed through a prior event of interchromosomal trans-splicing.Strikingly,DNA fragments containing the asAmp with an upstream recombined cryptic promoter and the corresponding exogenous asAmp transcripts have been detected in human cells.Our findings shed lights on the mechanism of human ACAT1 56-kDa isoform production,reveal an exogenous-endogenous trans-splicing system,in which recombinant plasmid-derived exogenous transcripts are linked with endogenous cellular RNAs in human cells,and suggest that exogenous DNA might affect human gene expression at both DNA and RNA levels.

  1. Are zinc-bound metallothionein isoforms (I+II and III involved in impaired thymulin production and thymic involution during ageing?

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    Cipriano Catia

    2004-11-01

    Full Text Available Abstract Background With advancing age, thymic efficiency shows progressive decline due to thymic involution allowing impaired cell-mediated immunity and the appearance of age-related diseases. The intrinsic cause of thymic involution is still undefined. Chronic inflammation and high glucocorticoids (GCs may be involved. However, transgenic mice, with increased GC sensitivity and over expression of GC receptors, display delayed age-associated thymic involution. This fact suggests that other substances may affect thymic involution. Among them, both isoforms of metallothioneins (MTs I+II and III are the major candidates because their increments leads to organ atrophy in constant stress and are induced by IL-6, which increases in ageing. Enhanced MTs in ageing allows constant sequester of zinc ions and no subsequent zinc release leading to low zinc ion bioavailability for thymic efficiency. This sequester is very limited in very old age. Thus, we have investigated the MTmRNA (I+II and III in the thymus from young, old and very old mice. Methods MTmRNA and IL-6mRNA (RT-PCR in the thymus from different donors were tested. Concomitantly, TECs proliferation, zinc ion bioavailability (ratio total thymulin/active thymulin, thymulin activity and corticosterone were tested from different donors. Results Both isoforms of MTmRNA and IL-6mRNA increase in old thymus coupled with low zinc ion bioavailability, reduced TECs proliferation, impaired thymulin activity and enhanced plasma corticosterone in comparison with young. Conversely, although the thymus is involuted in very old mice because of no changes in thymus weight in comparison to old mice, reduced MTmRNA, especially MT-I+II isoforms, and low IL6mRNA occur. Concomitantly, good zinc ion bioavailability, maintained TECs proliferation, satisfactory thymulin activity and reduced corticosterone are observed in very old mice. Conclusions The concomitant increments by high IL-6 of both MT isoforms in the

  2. Are zinc-bound metallothionein isoforms (I+II and III) involved in impaired thymulin production and thymic involution during ageing?

    Science.gov (United States)

    Mocchegiani, Eugenio; Giacconi, Robertina; Cipriano, Catia; Muti, Elisa; Gasparini, Nazzarena; Malavolta, Marco

    2004-11-12

    BACKGROUND: With advancing age, thymic efficiency shows progressive decline due to thymic involution allowing impaired cell-mediated immunity and the appearance of age-related diseases. The intrinsic cause of thymic involution is still undefined. Chronic inflammation and high glucocorticoids (GCs) may be involved. However, transgenic mice, with increased GC sensitivity and over expression of GC receptors, display delayed age-associated thymic involution. This fact suggests that other substances may affect thymic involution. Among them, both isoforms of metallothioneins (MTs) I+II and III are the major candidates because their increments leads to organ atrophy in constant stress and are induced by IL-6, which increases in ageing. Enhanced MTs in ageing allows constant sequester of zinc ions and no subsequent zinc release leading to low zinc ion bioavailability for thymic efficiency. This sequester is very limited in very old age. Thus, we have investigated the MTmRNA (I+II and III) in the thymus from young, old and very old mice. METHODS: MTmRNA and IL-6mRNA (RT-PCR) in the thymus from different donors were tested. Concomitantly, TECs proliferation, zinc ion bioavailability (ratio total thymulin/active thymulin), thymulin activity and corticosterone were tested from different donors. RESULTS: Both isoforms of MTmRNA and IL-6mRNA increase in old thymus coupled with low zinc ion bioavailability, reduced TECs proliferation, impaired thymulin activity and enhanced plasma corticosterone in comparison with young. Conversely, although the thymus is involuted in very old mice because of no changes in thymus weight in comparison to old mice, reduced MTmRNA, especially MT-I+II isoforms, and low IL6mRNA occur. Concomitantly, good zinc ion bioavailability, maintained TECs proliferation, satisfactory thymulin activity and reduced corticosterone are observed in very old mice. CONCLUSIONS: The concomitant increments by high IL-6 of both MT isoforms in the thymus from old mice may

  3. Expression and characterization of plasma membrane aquaporins in stomatal complexes of Zea mays.

    Science.gov (United States)

    Heinen, Robert B; Bienert, Gerd Patrick; Cohen, David; Chevalier, Adrien S; Uehlein, Norbert; Hachez, Charles; Kaldenhoff, Ralf; Le Thiec, Didier; Chaumont, François

    2014-10-01

    Stomata, the microscopic pores on the surface of the aerial parts of plants, are bordered by two specialized cells, known as guard cells, which control the stomatal aperture according to endogenous and environmental signals. Like most movements occurring in plants, the opening and closing of stomata are based on hydraulic forces. During opening, the activation of plasma membrane and tonoplast transporters results in solute accumulation in the guard cells. To re-establish the perturbed osmotic equilibrium, water follows the solutes into the cells, leading to their swelling. Numerous studies have contributed to the understanding of the mechanism and regulation of stomatal movements. However, despite the importance of transmembrane water flow during this process, only a few studies have provided evidence for the involvement of water channels, called aquaporins. Here, we microdissected Zea mays stomatal complexes and showed that members of the aquaporin plasma membrane intrinsic protein (PIP) subfamily are expressed in these complexes and that their mRNA expression generally follows a diurnal pattern. The substrate specificity of two of the expressed ZmPIPs, ZmPIP1;5 and ZmPIP1;6, was investigated by heterologous expression in Xenopus oocytes and yeast cells. Our data show that both isoforms facilitate transmembrane water diffusion in the presence of the ZmPIP2;1 isoform. In addition, both display CO2 permeability comparable to that of the CO2 diffusion facilitator NtAQP1. These data indicate that ZmPIPs may have various physiological roles in stomatal complexes.

  4. Sensitivity of Aquaporin-4 Isoforms Binding to Antibody in Neuromyelitis Optica%表达AQP4不同亚型的细胞系检测AQP4抗体敏感性的研究

    Institute of Scientific and Technical Information of China (English)

    刘俊秀; 赖蓉; 钟德霞; 黄帆; 丰岩清; 梁秀龄

    2012-01-01

    [目的]研究表达不同亚型的AQP4细胞系检测中国人群视神经脊髓炎患者血清中AQP4抗体的敏感性.[方法]根据纳入标准共获取血清205例,其中包括视神经脊髓炎40例,长节段性脊髓炎39例,复发性视神经炎39例,多发性硬化47例,另设40例健康对照.使用稳定表达水通道蛋白4的四种细胞系HEK293/pcDNA3.1(+)-M23-AQP4、HEK293/pcDNA3.1(+)-M1-AQP4、HEK293/pEGFP-N3-M23-AQP4、HEK293/pEGFP-N3-M1-AQP4通过细胞间接免疫荧光法分别检测以上血清中的AQP4抗体.[结果]NMO-IgG普遍存在于视神经脊髓炎疾病谱中.AQP4抗体同AQP4两种亚型结合敏感性比较发现,M23-AQP4的敏感性高于M1-AQP4.同时绿色荧光蛋白作为标签蛋白,可能干扰抗体同AQP4的结合.[结论]M23-AQP4作为靶抗原检测血清中AQP4抗体优于M1-AQP4,临床中采用细胞间接免疫荧光法对抗体进行检测时应首选表达M23-AQP4亚型的细胞系进行检测.%[Objective] To investigate the performance of AQP4 isoform to detect AQP4 antibodies in neuromyelitis optica(NMO) patient's sera. [Methods] We tested 205 masked serum samples from 40 NMO patients, 39 longitudinally extensive transverse myelitis (LETM) patients, 39 patients with recurrent optic neuritis (rON), 47 patients with multiple sclerosis (MS) and 40 healthy controls with the indirect immunofluorescence with a composite substrate of HEK293/pcDNA3.1 (+ )-M23-AQP4, HEK293/ pcDNA3.1(+)-Ml-AQP4, HEK293/pEGFP-N3-M23-AQP4, HEK293/pEGFP-N3-Ml- AQP4. [Results] We found antibody to AQP4 was present in NMO spectrums. M23-AQP4 was more sensitivity than the M1-AQP4 and while the enhanced green fluorescent protein (EGFP) which was a tag protein may be interfere with the combination between the AQP4 antibody and AQP4. [Conclusion] M23-AQP4 appears to have a higher sensitivity than the M1-AQP4. It is expected to be a first consideration for the detection AQP4 antibody with the cell based assay.

  5. Na+/H+ exchanger isoform 1 induced cardiomyocyte hypertrophy involves activation of p90 ribosomal s6 kinase.

    Science.gov (United States)

    Jaballah, Maiy; Mohamed, Iman A; Alemrayat, Bayan; Al-Sulaiti, Fatima; Mlih, Mohamed; Mraiche, Fatima

    2015-01-01

    Studies using pharmacological and genetic approaches have shown that increased activity/expression of the Na+/H+ exchanger isoform 1 (NHE1) play a critical role in the pathogenesis of cardiac hypertrophy. Despite the importance of NHE1 in cardiac hypertrophy, severe cerebrovascular side effects were associated with the use of NHE1 inhibitors when administered to patients with myocardial infarctions. p90 ribosomal S6 Kinase (RSK), a downstream regulator of the mitogen-activated protein kinase pathway, has also been implicated in cardiac hypertrophy. We hypothesized that RSK plays a role in the NHE1 induced cardiomyocyte hypertrophic response. Infection of H9c2 cardiomyoblasts with the active form of the NHE1 adenovirus induced hypertrophy and was associated with an increase in the phosphorylation of RSK (Phypertrophy such as cell area, protein content and atrial natriuretic mRNA expression were significantly reduced in H9c2 cardiomyoblasts infected with active NHE1 in the presence of dominant negative RSK (DN-RSK) (Phypertrophy is accounted for by increased activation and phosphorylation of RSK, which subsequently increased the phosphorylation of GATA4; eventually activating fetal gene transcriptional machinery.

  6. Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana.

    Science.gov (United States)

    Guénin, Stéphanie; Mareck, Alain; Rayon, Catherine; Lamour, Romain; Assoumou Ndong, Yves; Domon, Jean-Marc; Sénéchal, Fabien; Fournet, Françoise; Jamet, Elisabeth; Canut, Hervé; Percoco, Giuseppe; Mouille, Grégory; Rolland, Aurélia; Rustérucci, Christine; Guerineau, François; Van Wuytswinkel, Olivier; Gillet, Françoise; Driouich, Azeddine; Lerouge, Patrice; Gutierrez, Laurent; Pelloux, Jérôme

    2011-10-01

    • Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. • A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. • We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. • Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.

  7. Aquaporin-10 represents an alternative pathway for glycerol efflux from human adipocytes.

    Directory of Open Access Journals (Sweden)

    Umberto Laforenza

    Full Text Available BACKGROUND: Glycerol outflow from adipocytes has been considered for a decade to be mediated by aquaporin-7, an aquaglyceroporin highly expressed in the adipose tissue. Its involvement in glycerol metabolism has been widely studied also in humans. Recent studies in different aquaporin-7 KO mice models pose two different questions 1 the exact localization of aquaporin-7 in human white adipose tissue; 2 the existence of other aquaglyceroporins that work with aquaporin-7 to guarantee glycerol efflux and thus a normal adiposity in humans. To this purpose we investigated the expression, the localization and the functioning of aquaglyceroporin-10 in subcutaneous white adipose tissue, in isolated and cultured differentiated adipocytes. METHODOLOGY/PRINCIPAL FINDINGS: Aquaporin-7 and -10 were expressed in the white adipose tissue both at mRNA and at protein level. Immunofluorescence revealed aquaporin-7 and -10 labelling in the human adipose tissue both to the plasma membrane and to a thin rim of cytoplasm of adipocytes. Aquaporin-7, but not aquaporin-10, colocalized with the endothelial marker CD34. Human cultured differentiated adipocytes showed an aquaporin-7 and -10 labelling mainly in the cytoplasm and in the lipid droplets with insulin reinforcing the lipid droplets staining and isoproterenol inducing its translocation to the plasma membrane compartment. Water and glycerol permeability measurements using adipocytes and adipose membrane vesicles confirmed the presence of functioning aquaglyceroporins. Aquaporin-10 silencing in human differentiated adipocytes resulted in a 50% decrease of glycerol and osmotic water permeability. CONCLUSIONS/SIGNIFICANCE: The results indicate that aquaporin-7, differently from mice, is present in both adipocyte and capillary plasma membranes of human adipose tissue. Aquaporin-10, on the contrary, is expressed exclusively in the adipocytes. The expression of two aquaglyceroporins in human adipose tissue is

  8. A Gold Coordination Compound as a Chemical Probe to Unravel Aquaporin-7 Function

    NARCIS (Netherlands)

    Madeira, Ana; de Almeida, Andreia; de Graaf, Chris; Camps, Marta; Zorzano, Antonio; Moura, Teresa F; Casini, Angela; Soveral, Graça

    2014-01-01

    Aquaporins (AQPs) are membrane water/glycerol channels that are involved in many physiological functions. Aquaporin-based modulators are predicted to have potential utility in the treatment of several diseases, as well as chemical tools to assess AQPs function in biological systems. We recently repo

  9. Osmotic water transport in aquaporins

    DEFF Research Database (Denmark)

    Zeuthen, Thomas; Alsterfjord, Magnus; Beitz, Eric

    2013-01-01

    Abstract  We test a novel, stochastic model of osmotic water transport in aquaporins. A solute molecule present at the pore mouth can either be reflected or permeate the pore. We assume that only reflected solute molecules induce osmotic transport of water through the pore, while permeating solute...... molecules give rise to no water transport. Accordingly, the rate of water transport is proportional to the reflection coefficient σ, while the solute permeability, P(S), is proportional to 1 - σ. The model was tested in aquaporins heterologously expressed in Xenopus oocytes. A variety of aquaporin channel...... sizes and geometries were obtained with the two aquaporins AQP1 and AQP9 and mutant versions of these. Osmotic water transport was generated by adding 20 mM of a range of different-sized osmolytes to the outer solution. The osmotic water permeability and the reflection coefficient were measured...

  10. Effects of propofol on ammonium chloride-exposed astrocyte morphology and aquaporin-4 expression

    Institute of Scientific and Technical Information of China (English)

    Hanjian Chen; Caifei Pan; Peng Guo; Yueying Zheng; Shengmei Zhu

    2011-01-01

    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.

  11. Electron crystallography and aquaporins.

    Science.gov (United States)

    Schenk, Andreas D; Hite, Richard K; Engel, Andreas; Fujiyoshi, Yoshinori; Walz, Thomas

    2010-01-01

    Electron crystallography of two-dimensional (2D) crystals can provide information on the structure of membrane proteins at near-atomic resolution. Originally developed and used to determine the structure of bacteriorhodopsin (bR), electron crystallography has recently been applied to elucidate the structure of aquaporins (AQPs), a family of membrane proteins that form pores mostly for water but also other solutes. While electron crystallography has made major contributions to our understanding of the structure and function of AQPs, structural studies on AQPs, in turn, have fostered a number of technical developments in electron crystallography. In this contribution, we summarize the insights electron crystallography has provided into the biology of AQPs, and describe technical advancements in electron crystallography that were driven by structural studies on AQP 2D crystals. In addition, we discuss some of the lessons that were learned from electron crystallographic work on AQPs.

  12. Aquaporin water channels: molecular mechanisms for human diseases.

    Science.gov (United States)

    Agre, Peter; Kozono, David

    2003-11-27

    Although water is the major component of all biological fluids, the molecular pathways for water transport across cell membranes eluded identification until the discovery of the aquaporin family of water channels. The atomic structure of mammalian AQP1 illustrates how this family of proteins is freely permeated by water but not protons (hydronium ions, H3O+). Definition of the subcellular sites of expression predicted their physiological functions and potential clinical disorders. Analysis of several human disease states has confirmed that aquaporins are involved in multiple different illnesses including abnormalities of kidney function, loss of vision, onset of brain edema, starvation, and arsenic toxicity.

  13. Ammonia and urea permeability of mammalian aquaporins

    DEFF Research Database (Denmark)

    Litman, Thomas; Søgaard, Rikke; Zeuthen, Thomas

    2009-01-01

    The human aquaporins,AQP3,AQP7, AQP8,AQP9, and possibly AQP10, are permeable to ammonia, and AQP7, AQP9, and possibly AQP3, are permeable to urea. In humans, these aquaporins supplement the ammonia transport of the Rhesus (Rh) proteins and the urea transporters (UTs). The mechanism by which...... ammonium is transported by aquaporins is not fully resolved. A comparison of transport equations, models, and experimental data shows that ammonia is transported in its neutral form, NH(3). In the presence of NH(3), the aquaporin stimulates H(+) transport. Consequently, this transport of H(+) is only...... significant at alkaline pH. It is debated whether the H(+) ion passes via the aquaporin or by some external route; the investigation of this problem requires the aquaporin-expressing cell to be voltage-clamped. The ammonia-permeable aquaporins differ from other aquaporins by having a less restrictive aromatic...

  14. Pollen Aquaporins: The Solute Factor

    Science.gov (United States)

    Pérez Di Giorgio, Juliana A.; Soto, Gabriela C.; Muschietti, Jorge P.; Amodeo, Gabriela

    2016-01-01

    In the recent years, the biophysical properties and presumed physiological role of aquaporins (AQPs) have been expanded to specialized cells where water and solute exchange are crucial traits. Complex but unique processes such as stomatal movement or pollen hydration and germination have been addressed not only by identifying the specific AQP involved but also by studying how these proteins integrate and coordinate cellular activities and functions. In this review, we referred specifically to pollen-specific AQPs and analyzed what has been assumed in terms of transport properties and what has been found in terms of their physiological role. Unlike that in many other cells, the AQP machinery in mature pollen lacks plasma membrane intrinsic proteins, which are extensively studied for their high water capacity exchange. Instead, a variety of TIPs and NIPs are expressed in pollen. These findings have altered the initial understanding of AQPs and water exchange to consider specific and diverse solutes that might be critical to sustaining pollen’s success. The spatial and temporal distribution of the pollen AQPs also reflects a regulatory mechanism that allowing a properly adjusting water and solute exchange. PMID:27881985

  15. Water Channels Aquaporin 4 and -1 Expression in Subependymoma Depends on the Localization of the Tumors.

    Directory of Open Access Journals (Sweden)

    Susan Noell

    Full Text Available We analyzed aquaporin 4 and -1 expression in subependymomas, benign and slow growing brain tumors WHO grade I. Ten subependymoma cases were investigated, five of the fossa inferior and five of the fossa superior.Using immunohistochemistry, we observed different aquaporin expression patterns depending on localization: aquaporin 4 and -1 were detected in infratentorial subependymomas in the entire tumor tissue. In contrast, supratentorial subependymomas revealed aquaporin 4 and -1 expression only in border areas of the tumor. PCR analyses however showed no difference in aquaporin 4 expression between all subependymomas independent of localization but at higher levels than in normal brain. In contrast, aquaporin 1 RNA levels were found to be higher only in infratentorial samples compared to supratentorial and normal brain samples. The reason for the different distribution pattern of aquaporin 4 in subependymomas still remains unclear. On the cellular level, aquaporin 4 was redistributed on the surface of the tumor cells, and in freeze fracture replicas no orthogonal arrays of particles were found. This was similar to our previous findings in malignant glioblastomas. From these studies, we know that extracellular matrix molecules within the tumor like agrin and its receptor alpha-dystroglycan are involved in forming orthogonal arrays of particles. In subependymomas neither agrin nor alpha-dystroglycan were detected around blood vessels.Taken together, we show in this study that in the benign subependymomas aquaporins 1 and 4 are dramatically redistributed and upregulated. We speculate that extracellular environments of infra- and supratentorial subependymomas are different and lead to different distribution patterns of aquaporin 4 and -1.

  16. Screening of aquaporin 7 and aquaporin 8 expression in 35 organs using semi-quantified RT-PCR methods

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    AIM:Aquaporins (AQP) are very important for the water transport across cell membrane. There are at least 10 mammalian AQPs( aquaporins 0-9) distributed in various organs and different kinds of cells. Each AQP has a distinct organ distribution, and this distribution could be useful in presuming the biological function of the aquaporin. The aim of this study was to figure out the distribution of aquaporin 7 (AQP7) and aquaporin 8(AQP8).METHODS:Semi-quantified RT-PCR was employed in this research. The ratio of OD value of target gene products divided by which of control gene products was calculated. Among 35 organs, testis, epididymis, skin, muscle, rectum, lung, bronchus, lymph node, stomach, duodenum, jejunum, ileum, colon, pancreas, liver, gall bladder, spleen, mammary gland, uterus, placenta, tonsil, urinary bladder, thyroid came from normal area of removed samples during operation. cDNA library of Prostate, thymus, salivary gland, penis, carotiol artery, adrenal gland, occipital lobe of brain, temporal lobe of brain, frontal lobe of brain, parietal lobe of brain, mid brain, choroid plexus are purchased from OriGene biotechnique company.RESULTS:①AQP 7 mRNA was found in testis, muscle, gall bladder, carotiol artery, lymph node and adrenal gland, and maximum expression of AQP 7 was in testis.②AQP 8 mRNA was found in pancreas, testis, skin and colon. and maximum expression of AQP 8 was in pancreas.CONCLUSION:Coexistence of AQP 7 and 8 in testis was confirmed, which suggested that both of these two aquaporins were involved in the regulation of testis function.

  17. Aquaporin-4 and ischemic brain edema

    Institute of Scientific and Technical Information of China (English)

    Saihong Dun; Yang Guo

    2007-01-01

    OBJECTIVE: To investigate the relationship of aquaporin 4 (AQP4) and brain edema.DATA SOURCES: Using the terms of "aquaporin-4, brain edema", we searched PubMed database to identify studies published from January 1997 to April 2006 in the English languages. Meanwhile, we also searched China National Knowledge Infrastructure (CNKI) for related studies.STUDY SELECTION: The collected data were selected firstly. Studies on AQP4 and brain edema were chosen and their full-texts were searched for, and those with repetitive or review studies were excluded.DATA EXTRACTION: Totally 146 related studies were collected, 42 of them were involved and the other 104 studies were used for reading reference data.DATA SYNTHESIS: AQP4 is a selective water permeable integral membrane protein. It is mainly expressed in astrocytes and ependymocyte, and is the important structural basis for water regulation and transportation between glial cells and cerebrospinal fluid or vessels. Phosphorylation is involved in the regulation of AQP4.AQP4 participates in the formation of brain edema caused by various factors. Studies on the structure and pathological changes of AQP4 are still in the initial stage, and the role and mechanism of AQP4 in the formation of brain edema is very unclear.CONCLUSION: AQP4 plays a critical regulating role in the formation of ischemic brain edema, but whether it is regulated by drugs lacks reliable evidence.

  18. Tonoplast Aquaporins Facilitate Lateral Root Emergence.

    Science.gov (United States)

    Reinhardt, Hagen; Hachez, Charles; Bienert, Manuela Désirée; Beebo, Azeez; Swarup, Kamal; Voß, Ute; Bouhidel, Karim; Frigerio, Lorenzo; Schjoerring, Jan K; Bennett, Malcolm J; Chaumont, Francois

    2016-03-01

    Aquaporins (AQPs) are water channels allowing fast and passive diffusion of water across cell membranes. It was hypothesized that AQPs contribute to cell elongation processes by allowing water influx across the plasma membrane and the tonoplast to maintain adequate turgor pressure. Here, we report that, in Arabidopsis (Arabidopsis thaliana), the highly abundant tonoplast AQP isoforms AtTIP1;1, AtTIP1;2, and AtTIP2;1 facilitate the emergence of new lateral root primordia (LRPs). The number of lateral roots was strongly reduced in the triple tip mutant, whereas the single, double, and triple tip mutants showed no or minor reduction in growth of the main root. This phenotype was due to the retardation of LRP emergence. Live cell imaging revealed that tight spatiotemporal control of TIP abundance in the tonoplast of the different LRP cells is pivotal to mediating this developmental process. While lateral root emergence is correlated to a reduction of AtTIP1;1 and AtTIP1;2 protein levels in LRPs, expression of AtTIP2;1 is specifically needed in a restricted cell population at the base, then later at the flanks, of developing LRPs. Interestingly, the LRP emergence phenotype of the triple tip mutants could be fully rescued by expressing AtTIP2;1 under its native promoter. We conclude that TIP isoforms allow the spatial and temporal fine-tuning of cellular water transport, which is critically required during the highly regulated process of LRP morphogenesis and emergence.

  19. Aquaporins of the PIP2 Class Are Required for Efficient Anther Dehiscence in Tobacco

    Science.gov (United States)

    Bots, Marc; Vergeldt, Frank; Wolters-Arts, Mieke; Weterings, Koen; van As, Henk; Mariani, Celestina

    2005-01-01

    Several processes during sexual reproduction in higher plants involve the movement of water between cells or tissues. Before flower anthesis, anther and pollen dehydration takes place before the release of mature pollen at dehiscence. Aquaporins represent a class of proteins that mediates the movement of water over cellular membranes. Aquaporins of the plasmamembrane PIP2 family are expressed in tobacco (Nicotiana tabacum) anthers and may therefore be involved in the movement of water in this organ. To gain more insight into the role these proteins may play in this process, we have analyzed their localization using immunolocalizations and generated plants displaying RNA interference of PIP2 aquaporins. Our results indicate that PIP2 protein expression is modulated during anther development. Furthermore, in tobacco PIP2 RNA interference plants, anther dehydration was slower, and dehiscence occurred later when compared with control plants. Together, our results suggest that aquaporins of the PIP2 class are required for efficient anther dehydration prior to dehiscence. PMID:15734911

  20. Identification of cytochrome P450 isoforms involved in the metabolism of paroxetine and estimation of their importance for human paroxetine metabolism using a population-based simulator

    DEFF Research Database (Denmark)

    Jornil, Jakob; Jensen, Klaus Gjervig; Larsen, Frank

    2010-01-01

    the importance of the identified paroxetine-metabolizing P450 isoforms for human metabolism, taking mechanism-based inhibition into account. The amount of active hepatic CYP2D6 and CYP3A4 (not inactivated by mechanism-based inhibition) was also estimated by Simcyp. For extensive and poor metabolizers of CYP2D6......We identify here for the first time the low-affinity cytochrome P450 (P450) isoforms that metabolize paroxetine, using cDNA-expressed human P450s measuring substrate depletion and paroxetine-catechol (product) formation by liquid chromatography-tandem mass spectrometry. CYP1A2, CYP2C19, CYP2D6, CYP......3A4, and CYP3A5 were identified as paroxetine-catechol-forming P450 isoforms, and CYP2C19 and CYP2D6 were identified as metabolizing P450 isoforms by substrate depletion. Michaelis-Menten constants K(m) and V(max) were determined by product formation and substrate depletion. Using selective...

  1. In vitro characterization of the cytochrome P450 isoforms involved in the metabolism of 6-methoxy-2-napthylacetic acid, an active metabolite of the prodrug nabumetone.

    Science.gov (United States)

    Matsumoto, Kaori; Nemoto, Eiichi; Hasegawa, Tetsuya; Akimoto, Masayuki; Sugibayashi, Kenji

    2011-01-01

    The cytochrome P450 (CYP) isoforms that catalyze the oxidation metabolism of 6-methoxy-2-napthylacetic acid (6-MNA), an active metabolite of nabumetone, were studied in rats and humans. Using an extractive reversed-phase HPLC assay with fluorescence detection, monophasic Michaelis-Menten kinetics was obtained for the formation of 6-hydroxy-2-naphthylacetic acid (6-HNA) in liver microsomes of rats and humans, and kinetic analysis showed that the K(m) and V(max) values for the formation of 6-HNA in humans and rats were 640.0 ± 30.9 and 722.9 ± 111.7 µM, and 1167.5 ± 33.0 and 1312.7 ± 73.8 pmol min⁻¹ mg protein⁻¹, respectively. The CYPs responsible for metabolism of 6-MNA in liver microsomes of rats and humans were identified using correlation study, recombinant CYP supersomes, and specific CYP inhibitors and antibodies. Recombinant human CYP2C9 exhibited appreciable catalytic activity with respect to 6-HNA formation from 6-MNA. Among 14 recombinant rat CYPs examined, CYP2C6, CYP2C11 and CYP1A2 were involved in the metabolism of 6-MNA. Sulfaphenazole (a selective inhibitor of CYP2C9) inhibited the formation of 6-HNA in pooled human microsomes by 89%, but failed to inhibit this reaction in rat liver microsomes. The treatment of pooled human liver microsomes with an antibody against CYP2C9 inhibited the formation of 6-HNA by about 80%. The antibody against CYP2C11 suppressed the activity by 20 to 30% in rat microsomes, whereas that of CYP1A2 microsomes did not show drastic inhibition. These findings suggest that CYP2C9 has the highest catalytic activity of 6-MNA metabolism in humans. In contrast, metabolism of 6-MNA is suggested to be mediated mainly by CYP2C6 and CYP2C11 in rats.

  2. Aquaporins are upregulated in glandular epithelium at the time of implantation in the rat.

    Science.gov (United States)

    Lindsay, Laura A; Murphy, Christopher R

    2007-03-01

    Regulation of luminal fluid is essential for blastocyst implantation. While it has been known for quite some time that there is a reduction in the amount of luminal fluid at the time of implantation, the mechanisms regulating this process are only just emerging. Previous studies have shown an upregulation of aquaporin (AQP) 5 channels in luminal epithelial cells at the time of implantation providing a mechanism for fluid reabsorption across the surface epithelium. However to date the contribution of fluid reabsorption by glandular epithelial cells has not been established. This study using reverse transcriptase polymerase chain reaction demonstrates the presence of several AQP isoforms in the rat uterus at the time of implantation while immunofluorescence data demonstrates an apical distribution of AQPs5 and 9 in the glandular epithelium at the time of implantation. The presence of AQPs5 and 9 in the apical plasma membrane of the glandular epithelium seen in this study provides a mechanism for transcellular fluid transport across these glandular epithelial cells similar to that seen in luminal epithelial cells. The reabsorption of glandular fluid via AQP channels may also regulate luminal fluid volume and be involved in the reduction in luminal fluid seen at the time of implantation.

  3. Aquaporins in desert rodent physiology.

    Science.gov (United States)

    Pannabecker, Thomas L

    2015-08-01

    Desert rodents face a sizeable challenge in maintaining salt and water homeostasis due to their life in an arid environment. A number of their organ systems exhibit functional characteristics that limit water loss above that which occurs in non-desert species under similar conditions. These systems include renal, pulmonary, gastrointestinal, nasal, and skin epithelia. The desert rodent kidney preserves body water by producing a highly concentrated urine that reaches a maximum osmolality nearly three times that of the common laboratory rat. The precise mechanism by which urine is concentrated in any mammal is unknown. Insights into the process may be more apparent in species that produce highly concentrated urine. Aquaporin water channels play a fundamental role in water transport in several desert rodent organ systems. The role of aquaporins in facilitating highly effective water preservation in desert rodents is only beginning to be explored. The organ systems of desert rodents and their associated AQPs are described.

  4. Renal aquaporins and water balance disorders

    DEFF Research Database (Denmark)

    Kortenoeven, Marleen; Fenton, Robert A.

    2013-01-01

    associated with disturbed water homeostasis. Hyponatremia with increased AQP levels can be caused by diseases with low effective circulating blood volume, such as congestive heart failure, or osmoregulation disorders such as the syndrome of inappropriate secretion of antidiuretic hormone. Treatment consists......BACKGROUND: Aquaporins (AQPs) are a family of proteins that can act as water channels. Regulation of AQPs is critical to osmoregulation and the maintenance of body water homeostasis. Eight AQPs are expressed in the kidney of which five have been shown to play a role in body water balance; AQP1, AQP......2, AQP3, AQP4 and AQP7. AQP2 in particular is regulated by vasopressin. SCOPE OF REVIEW: This review summarizes our current knowledge of the underlying mechanisms of various water balance disorders and their treatment strategies. MAJOR CONCLUSIONS: Dysfunctions of AQPs are involved in disorders...

  5. [Roles of Aquaporins in Brain Disorders].

    Science.gov (United States)

    Yasui, Masato

    2015-06-01

    Aquaporin (AQP) is a water channel protein that is expressed in the cell membranes. AQPs are related to several kinds of human diseases such as cataract. In the mammalian central nervous system (CNS), AQP4 is specifically expressed in the astrocyte membranes lining the perivascular and periventricular structures. AQP4 plays a role in the development of brain edema associated with certain brain disorders. Neuromyelitis optica (NMO) is a demyelinating disorder, and patients with NMO develop autoimmune antibodies against AQP4 in their serum. Therefore, AQP4 is involved in NMO pathogenesis. A new concept referred to as "glymphatic pathway" has been recently proposed to explain the lymphatic system in the CNS. Dysfunction of the "glymphatic pathway" may cause several neurodegenerative diseases and mood disorders. Importantly, AQP4 may play a role in the "glymphatic pathway". Further investigation of AQP4 in CNS disorders is necessary, and a new drug against AQP4 is expected.

  6. Aquaporin 1, a potential therapeutic target for migraine with aura

    Directory of Open Access Journals (Sweden)

    Jiang Xinghong

    2010-10-01

    Full Text Available Abstract The pathophysiology of migraine remains largely unknown. However, evidence regarding the molecules participating in the pathophysiology of migraine has been accumulating. Water channel proteins, known as aquaporins (AQPs, notably AQP-1 and AQP-4, appears to be involved in the pathophysiology of several neurological diseases. This review outlines newly emerging evidence indicating that AQP-1 plays an important role in pain signal transduction and migraine and could therefore serve as a potential therapeutic target for these diseases.

  7. Robust High Performance Aquaporin based Biomimetic Membranes

    DEFF Research Database (Denmark)

    Helix Nielsen, Claus; Zhao, Yichun; Qiu, C.;

    2013-01-01

    on top of a support membrane. Control membranes, either without aquaporins or with the inactive AqpZ R189A mutant aquaporin served as controls. The separation performance of the membranes was evaluated by cross-flow forward osmosis (FO) and reverse osmosis (RO) tests. In RO the ABM achieved a water...

  8. Aquaporin-4: orthogonal array assembly, CNS functions, and role in neuromyelitis optica

    Institute of Scientific and Technical Information of China (English)

    Alan S VERKMAN; Julien RATELADE; Andrea ROSSI; Hua ZHANG; Lukmanee TRADTRANTIP

    2011-01-01

    Aquaporin-4 (AQP4) is a water-selective transporter expressed in astrocytes throughout the central nervous system, as well as in kidney, lung, stomach and skeletal muscle. The two AQP4 isoforms produced by alternative spicing, M1 and M23 AQP4, form heterotetramers that assemble in cell plasma membranes in supramolecular structures called orthogonal arrays of particles (OAPs).Phenotype analysis of AQP4-null mice indicates the involvement of AQP4 in brain and spinal cord water balance, astrocyte migration, neural signal transduction and neuroinflammation. AQP4-null mice manifest reduced brain swelling in cytotoxic cerebral edema, but increased brain swelling in vasogenic edema and hydrocephalus. AQP4 deficiency also increases seizure duration,impairs glial scarring, and reduces the severity of autoimmune neuroinflammation. Each of these phenotypes is likely explicable on the basis of reduced astrocyte water permeability in AQP4 deficiency. AQP4 is also involved in the neuroinflammatory demyelinating disease neuromyelitis optica (NMO), where autoantibodies (NMO-lgG) targeting AQP4 produce astrocyte damage and inflammation.Mice administered NMO-lgG and human complement by intracerebral injection develop characteristic NMO lesions with neuroinflammation, demyelination, perivascular complement deposition and loss of glial fibrillary acidic protein and AQP4 immunoreactivity.Our findings suggest the potential utility of AQP4-based therapeutics, including small-molecule modulators of AQP4 water transport function for therapy of brain swelling, injury and epilepsy, as well as small-molecule or monoclonal antibody blockers of NMO-lgG binding to AQP4 for therapy of NMO.

  9. Aquaporin 4 expression and ultrastructure of the blood-brain barrier following cerebral contusion injury

    Institute of Scientific and Technical Information of China (English)

    Xinjun Li; Yangyun Han; Hong Xu; Zhongshu Sun; Zengjun Zhou; Xiaodong Long; Yumin Yang; Linbo Zou

    2013-01-01

    This study aimed to investigate aquaporin 4 expression and the ultrastructure of the blood-brain barrier at 2–72 hours following cerebral contusion injury, and correlate these changes to the formation of brain edema. Results revealed that at 2 hours after cerebral contusion and laceration injury, aquaporin 4 expression significantly increased, brain water content and blood-brain barrier permeability increased, and the number of pinocytotic vesicles in cerebral microvascular endothelial cells increased. In addition, the mitochondrial accumulation was observed. As contusion and laceration injury became aggravated, aquaporin 4 expression continued to increase, brain water content and blood-brain barrier permeability gradually increased, brain capillary endothelial cells and astrocytes swelled, and capillary basement membrane injury gradually increased. The above changes were most apparent at 12 hours after injury, after which they gradually attenuated. Aquaporin 4 expression positively correlated with brain water content and the blood-brain barrier index. Our experimental findings indicate that increasing aquaporin 4 expression and blood-brain barrier permeability after cerebral contusion and laceration injury in humans is involved in the formation of brain edema.

  10. Role of Aquaporin 0 in lens biomechanics.

    Science.gov (United States)

    Sindhu Kumari, S; Gupta, Neha; Shiels, Alan; FitzGerald, Paul G; Menon, Anil G; Mathias, Richard T; Varadaraj, Kulandaiappan

    2015-07-10

    fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia.

  11. Aquaporin 5 Interacts with Fluoride and Possibly Protects against Caries.

    Directory of Open Access Journals (Sweden)

    Ida Anjomshoaa

    Full Text Available Aquaporins (AQP are water channel proteins and the genes coding for AQP2, AQP5, and AQP6 are clustered in 12q13. Since AQP5 is expressed in serous acinar cells of salivary glands, we investigated its involvement in caries. DNA samples from 1,383 individuals from six groups were studied. Genotypes of eight single nucleotide polymorphisms covering the aquaporin locus were tested for association with caries experience. Interaction with genes involved in enamel formation was tested. The association between enamel microhardness at baseline, after creation of artificial caries lesion, and after exposure to fluoride and the genetic markers in AQP5 was tested. Finally, AQP5 expression in human whole saliva, after exposure to fluoride in a mammary gland cell line, which is known to express AQP5, and in Wistar rats was also verified. Nominal associations were found between caries experience and markers in the AQP5 locus. Since these associations suggested that AQP5 may be inhibited by levels of fluoride in the drinking water that cause fluorosis, we showed that fluoride levels above optimal levels change AQP5 expression in humans, cell lines, and rats. We have shown that AQP5 is involved in the pathogenesis of caries and likely interacts with fluoride.

  12. Aquaporins as potential drug targets

    Institute of Scientific and Technical Information of China (English)

    Fang WANG; Xue-chao FENG; Yong-ming LI; Hong YANG; Tong-hui MA

    2006-01-01

    The aquaporins (AQP) are a family of integral membrane proteins that selectively transport water and,in some cases,small neutral solutes such as glycerol and urea.Thirteen mammalian AQP have been molecularly identified and localized to various epithelial,endothelial and other tissues.Phenotype studies of transgenic mouse models of AQP knockout,mutation,and in some cases humans with AQP mutations have demonstrated essential roles for AQP in mammalian physiology and pathophysiology,including urinary concentrating function,exocrine glandular fluid secretion,brain edema formation,regulation of intracranial and intraocular pressure,skin hydration,fat metabolism,tumor angiogenesis and cell migration.These studies suggest that AQP may be potential drug targets for not only new diuretic reagents for various forms of pathological water retention,but also targets for novel therapy of brain edema,inflammatory disease,glaucoma,obesity,and cancer.However,potent AQP modulators for in vivo application remain to be discovered.

  13. Expression of Fragaria vesca PIP aquaporins in response to drought stress: PIP down-regulation correlates with the decline in substrate moisture content.

    Science.gov (United States)

    Šurbanovski, Nada; Sargent, Daniel J; Else, Mark A; Simpson, David W; Zhang, Hanma; Grant, Olga M

    2013-01-01

    PIP aquaporin responses to drought stress can vary considerably depending on the isoform, tissue, species or level of stress; however, a general down-regulation of these genes is thought to help reduce water loss and prevent backflow of water to the drying soil. It has been suggested therefore, that it may be necessary for the plant to limit aquaporin production during drought stress, but it is unknown whether aquaporin down-regulation is gradual or triggered by a particular intensity of the stress. In this study, ten Fragaria PIP genes were identified from the woodland strawberry (Fragaria vesca L.) genome sequence and characterised at the sequence level. The water relations of F. vesca were investigated and the effect of different intensities of drought stress on the expression of four PIP genes, as well as how drought stress influences their diurnal transcription was determined. PIP down-regulation in the root corresponded to the level of drought stress. Moreover, transcript abundance of two genes highly expressed in the root (FvPIP1;1 and FvPIP2;1) was strongly correlated to the decline in substrate moisture content. The amplitude of diurnal aquaporin expression in the leaves was down-regulated by drought without altering the pattern, but showing an intensity-dependent effect. The results show that transcription of PIP aquaporins can be fine-tuned with the environment in response to declining water availability.

  14. Expression of Fragaria vesca PIP aquaporins in response to drought stress: PIP down-regulation correlates with the decline in substrate moisture content.

    Directory of Open Access Journals (Sweden)

    Nada Šurbanovski

    Full Text Available PIP aquaporin responses to drought stress can vary considerably depending on the isoform, tissue, species or level of stress; however, a general down-regulation of these genes is thought to help reduce water loss and prevent backflow of water to the drying soil. It has been suggested therefore, that it may be necessary for the plant to limit aquaporin production during drought stress, but it is unknown whether aquaporin down-regulation is gradual or triggered by a particular intensity of the stress. In this study, ten Fragaria PIP genes were identified from the woodland strawberry (Fragaria vesca L. genome sequence and characterised at the sequence level. The water relations of F. vesca were investigated and the effect of different intensities of drought stress on the expression of four PIP genes, as well as how drought stress influences their diurnal transcription was determined. PIP down-regulation in the root corresponded to the level of drought stress. Moreover, transcript abundance of two genes highly expressed in the root (FvPIP1;1 and FvPIP2;1 was strongly correlated to the decline in substrate moisture content. The amplitude of diurnal aquaporin expression in the leaves was down-regulated by drought without altering the pattern, but showing an intensity-dependent effect. The results show that transcription of PIP aquaporins can be fine-tuned with the environment in response to declining water availability.

  15. Identification and role of plasma membrane aquaporin in maize root

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using antiserum against expressed aquaporin fusion protein, GST-RD28, the distribution of aquaporin in the plasma membrane of maize root protoplasts has been examined under confocal laser scanning microscopy by indirect fluorescence staining. Results indicate that there are abundant aquaporins in maize roots, which are distributed in plasma membrane unevenly. Western blotting analysis of total protein solubilized from maize root plasma membrane shows that antiserum against GST-RD28 can cross-react with one protein around 55 ku. Another 28 ku protein can also be detected when the concentration of SDS and DTT in SDS-PAGE sample buffer is increased. The 55 and 28 ku proteins may be dimeric and monomeric of aquaporin respectively. Functional experiments show that aquaporin blocker HgCl2 and aquaporin antiserum can suppress the swelling of maize root protoplasts in hypotonic solution, indicating that aquaporin in plasma membrane of protoplast facilitates rapid transmembrane water flow.

  16. Desalination by biomimetic aquaporin membranes: Review of status and prospects

    DEFF Research Database (Denmark)

    Tang, C.Y.; Zhao, Y.; Wang, R.;

    2013-01-01

    Based on their unique combination of offering high water permeability and high solute rejection aquaporin proteins have attracted considerable interest over the last years as functional building blocks of biomimetic membranes for water desalination and reuse. The purpose of this review is to prov......Based on their unique combination of offering high water permeability and high solute rejection aquaporin proteins have attracted considerable interest over the last years as functional building blocks of biomimetic membranes for water desalination and reuse. The purpose of this review...... is to provide an overview of the properties of aquaporins, their preparation and characterization. We discuss the challenges in exploiting the remarkable properties of aquaporin proteins for membrane separation processes and we present various attempts to construct aquaporin in membranes for desalination......; including an overview of our own recent developments in aquaporin-based membranes. Finally we outline future prospects of aquaporin based biomimetic membrane for desalination and water reuse....

  17. Aquaporin, forward osmosis and biomimetic membranes.

    Science.gov (United States)

    Kocherginsky, Nikolai

    2013-12-01

    Aquaporin attracted attention not only of physiologists and biophysicists, but also of chemical engineers. Here we critically analyze a paper describing aquaporin-based artificial membranes, suggested for forward osmosis-based water purification (Wang et al. 2012, Small 8, pp. 1185-1190). Related papers published later by the same group are also discussed. We indicate recently developed general approach to describe membrane transport, membrane permeability and selectivity, which is applicable for forward osmosis. In addition, we also mention our papers describing simple nitrocellulose-based membranes, which have selective aqueous channels without proteins, but successfully imitate many properties of biomembranes.

  18. Tonoplast Aquaporins Facilitate Lateral Root Emergence1[OPEN

    Science.gov (United States)

    Hachez, Charles; Bienert, Manuela Désirée; Beebo, Azeez; Swarup, Kamal

    2016-01-01

    Aquaporins (AQPs) are water channels allowing fast and passive diffusion of water across cell membranes. It was hypothesized that AQPs contribute to cell elongation processes by allowing water influx across the plasma membrane and the tonoplast to maintain adequate turgor pressure. Here, we report that, in Arabidopsis (Arabidopsis thaliana), the highly abundant tonoplast AQP isoforms AtTIP1;1, AtTIP1;2, and AtTIP2;1 facilitate the emergence of new lateral root primordia (LRPs). The number of lateral roots was strongly reduced in the triple tip mutant, whereas the single, double, and triple tip mutants showed no or minor reduction in growth of the main root. This phenotype was due to the retardation of LRP emergence. Live cell imaging revealed that tight spatiotemporal control of TIP abundance in the tonoplast of the different LRP cells is pivotal to mediating this developmental process. While lateral root emergence is correlated to a reduction of AtTIP1;1 and AtTIP1;2 protein levels in LRPs, expression of AtTIP2;1 is specifically needed in a restricted cell population at the base, then later at the flanks, of developing LRPs. Interestingly, the LRP emergence phenotype of the triple tip mutants could be fully rescued by expressing AtTIP2;1 under its native promoter. We conclude that TIP isoforms allow the spatial and temporal fine-tuning of cellular water transport, which is critically required during the highly regulated process of LRP morphogenesis and emergence. PMID:26802038

  19. [Aquaporins in gametogenesis of vertebrate animals].

    Science.gov (United States)

    Skoblina, M N

    2008-01-01

    A review of the data on the presence, localization, and supposed role of aquaporin water channels in oocytes of Xenopus laevis, oogenesis and maturation of teleosts Sparus auratus and Oncorhynchus mykiss, oogenesis and oocyte maturation of rats and mice, and spermatogenesis of several mammalians.

  20. Fragment Screening of Human Aquaporin 1

    Directory of Open Access Journals (Sweden)

    Janet To

    2016-03-01

    Full Text Available Aquaporins (AQPs are membrane proteins that enable water transport across cellular plasma membranes in response to osmotic gradients. Phenotypic analyses have revealed important physiological roles for AQPs, and the potential for AQP water channel modulators in various disease states has been proposed. For example, AQP1 is overexpressed in tumor microvessels, and this correlates with higher metastatic potential and aggressiveness of the malignancy. Chemical modulators would help in identifying the precise contribution of water channel activity in these disease states. These inhibitors would also be important therapeutically, e.g., in anti-cancer treatment. This perceived importance contrasts with the lack of success of high-throughput screens (HTS to identify effective and specific inhibitors of aquaporins. In this paper, we have screened a library of 1500 “fragments”, i.e., smaller than molecules used in HTS, against human aquaporin (hAQP1 using a thermal shift assay and surface plasmon resonance. Although these fragments may not inhibit their protein target, they bound to and stabilized hAQP1 (sub mM binding affinities (KD, with an temperature of aggregation shift ΔTagg of +4 to +50 °C in a concentration-dependent fashion. Chemically expanded versions of these fragments should follow the determination of their binding site on the aquaporin surface.

  1. Role of Aquaporin 0 in lens biomechanics

    Energy Technology Data Exchange (ETDEWEB)

    Sindhu Kumari, S.; Gupta, Neha [Physiology and Biophysics, Stony Brook University, Stony Brook, NY (United States); Shiels, Alan [Washington University School of Medicine, St. Louis, MO (United States); FitzGerald, Paul G. [Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, CA (United States); Menon, Anil G. [University of Cincinnati College of Medicine, Cincinnati, OH (United States); Mathias, Richard T. [Physiology and Biophysics, Stony Brook University, Stony Brook, NY (United States); SUNY Eye Institute, NY (United States); Varadaraj, Kulandaiappan, E-mail: kulandaiappan.varadaraj@stonybrook.edu [Physiology and Biophysics, Stony Brook University, Stony Brook, NY (United States); SUNY Eye Institute, NY (United States)

    2015-07-10

    together they help to confer fiber cell shape, architecture and integrity. To our knowledge, this is the first report identifying the involvement of an aquaporin in lens biomechanics. Since accommodation is required in human lenses for proper focusing, alteration in the adhesion and/or water channel functions of AQP0 could contribute to presbyopia. - Highlights: • AQP0 aids in lens biomechanics. • AQP0 provides lens stiffness. • AQP0 is critical for lens transparency. • AQP0 could play a significant role in lens accommodation in human. • Alteration in the function(s) of lens AQP0 could lead to presbyopia.

  2. The Aquaporin Channel Repertoire of the Tardigrade Milnesium tardigradum.

    Science.gov (United States)

    Grohme, Markus A; Mali, Brahim; Wełnicz, Weronika; Michel, Stephanie; Schill, Ralph O; Frohme, Marcus

    2013-01-01

    Limno-terrestrial tardigrades are small invertebrates that are subjected to periodic drought of their micro-environment. They have evolved to cope with these unfavorable conditions by anhydrobiosis, an ametabolic state of low cellular water. During drying and rehydration, tardigrades go through drastic changes in cellular water content. By our transcriptome sequencing effort of the limno-terrestrial tardigrade Milnesium tardigradum and by a combination of cloning and targeted sequence assembly, we identified transcripts encoding eleven putative aquaporins. Analysis of these sequences proposed 2 classical aquaporins, 8 aquaglyceroporins and a single potentially intracellular unorthodox aquaporin. Using quantitative real-time PCR we analyzed aquaporin transcript expression in the anhydrobiotic context. We have identified additional unorthodox aquaporins in various insect genomes and have identified a novel common conserved structural feature in these proteins. Analysis of the genomic organization of insect aquaporin genes revealed several conserved gene clusters.

  3. Evidence of Positive Selection of Aquaporins Genes from Pontoporia blainvillei during the Evolutionary Process of Cetaceans.

    Directory of Open Access Journals (Sweden)

    Simone Lima São Pedro

    Full Text Available Marine mammals are well adapted to their hyperosmotic environment. Several morphological and physiological adaptations for water conservation and salt excretion are known to be present in cetaceans, being responsible for regulating salt balance. However, most previous studies have focused on the unique renal physiology of marine mammals, but the molecular bases of these mechanisms remain poorly explored. Many genes have been identified to be involved in osmotic regulation, including the aquaporins. Considering that aquaporin genes were potentially subject to strong selective pressure, the aim of this study was to analyze the molecular evolution of seven aquaporin genes (AQP1, AQP2, AQP3, AQP4, AQP6, AQP7, and AQP9 comparing the lineages of cetaceans and terrestrial mammals.Our results demonstrated strong positive selection in cetacean-specific lineages acting only in the gene for AQP2 (amino acids 23, 83, 107,179, 180, 181, 182, whereas no selection was observed in terrestrial mammalian lineages. We also analyzed the changes in the 3D structure of the aquaporin 2 protein. Signs of strong positive selection in AQP2 sites 179, 180, 181, and 182 were unexpectedly identified only in the baiji lineage, which was the only river dolphin examined in this study. Positive selection in aquaporins AQP1 (45, AQP4 (74, AQP7 (342, 343, 356 was detected in cetaceans and artiodactyls, suggesting that these events are not related to maintaining water and electrolyte homeostasis in seawater.Our results suggest that the AQP2 gene might reflect different selective pressures in maintaining water balance in cetaceans, contributing to the passage from the terrestrial environment to the aquatic. Further studies are necessary, especially those including other freshwater dolphins, who exhibit osmoregulatory mechanisms different from those of marine cetaceans for the same essential task of maintaining serum electrolyte balance.

  4. Neuromyelitis optica pathogenesis and aquaporin 4

    Directory of Open Access Journals (Sweden)

    Kerr Douglas

    2008-05-01

    Full Text Available Abstract Neuromyelitis optica (NMO is a severe, debilitating human disease that predominantly features immunopathology in the optic nerves and the spinal cord. An IgG1 autoantibody (NMO-IgG that binds aquaporin 4 (AQP4 has been identified in the sera of a significant number of NMO patients, as well as in patients with two related neurologic conditions, bilateral optic neuritis (ON, and longitudinal extensive transverse myelitis (LETM, that are generally considered to lie within the NMO spectrum of diseases. NMO-IgG is not the only autoantibody found in NMO patient sera, but the correlation of pathology in central nervous system (CNS with tissues that normally express high levels of AQP4 suggests NMO-IgG might be pathogenic. If this is the case, it is important to identify and understand the mechanism(s whereby an immune response is induced against AQP4. This review focuses on open questions about the "events" that need to be understood to determine if AQP4 and NMO-IgG are involved in the pathogenesis of NMO. These questions include: 1 How might AQP4-specific T and B cells be primed by either CNS AQP4 or peripheral pools of AQP4? 2 Do the different AQP4-expressing tissues and perhaps the membrane structural organization of AQP4 influence NMO-IgG binding efficacy and thus pathogenesis? 3 Does prior infection, genetic predisposition, or underlying immune dysregulation contribute to a confluence of events which lead to NMO in select individuals? A small animal model of NMO is essential to demonstrate whether AQP4 is indeed the incipient autoantigen capable of inducing NMO-IgG formation and NMO. If the NMO model is consistent with the human disease, it can be used to examine how changes in AQP4 expression and blood-brain barrier (BBB integrity, both of which can be regulated by CNS inflammation, contribute to inductive events for anti-AQP4-specific immune response. In this review, we identify reagents and experimental questions that need to be

  5. Expression and function of aquaporins in peripheral nervous system

    Institute of Scientific and Technical Information of China (English)

    Tong-hui MA; Hong-wen GAO; Xue-dong FANG; Hong YANG

    2011-01-01

    The expression and role of the aquaporin (AQP) family water channels in the peripheral nervous system was less investigated. Since 2004, however, significant progress has been made in the immunolocalization, regulation and function of AQPs in the peripheral nervous system. These studies showed selective localization of three AQPs (AQP1, AQP2, and AQP4) in dorsal root ganglion neurons,enteric neurons and glial cells, periodontal Ruffini endings, trigeminal ganglion neurons and vomeronasal sensory neurons. Functional characterization in transgenic knockout mouse model revealed important role of AQP1 in pain perception. This review will summarize the progress in this field and discuss possible involvement of AQPs in peripheral neuropathies and their potential as novel drug targets.

  6. The Hevea brasiliensis XIP aquaporin subfamily: genomic, structural and functional characterizations with relevance to intensive latex harvesting.

    Science.gov (United States)

    Lopez, David; Amira, Maroua Ben; Brown, Daniel; Muries, Beatriz; Brunel-Michac, Nicole; Bourgerie, Sylvain; Porcheron, Benoit; Lemoine, Remi; Chrestin, Hervé; Mollison, Ewan; Di Cola, Alessandra; Frigerio, Lorenzo; Julien, Jean-Louis; Gousset-Dupont, Aurélie; Fumanal, Boris; Label, Philippe; Pujade-Renaud, Valérie; Auguin, Daniel; Venisse, Jean-Stéphane

    2016-07-01

    X-Intrinsic Proteins (XIP) were recently identified in a narrow range of plants as a full clade within the aquaporins. These channels reportedly facilitate the transport of a wide range of hydrophobic solutes. The functional roles of XIP in planta remain poorly identified. In this study, we found three XIP genes (HbXIP1;1, HbXIP2;1 and HbXIP3;1) in the Hevea brasiliensis genome. Comprehensive bioinformatics, biochemical and structural analyses were used to acquire a better understanding of this AQP subfamily. Phylogenetic analysis revealed that HbXIPs clustered into two major groups, each distributed in a specific lineage of the order Malpighiales. Tissue-specific expression profiles showed that only HbXIP2;1 was expressed in all the vegetative tissues tested (leaves, stem, bark, xylem and latex), suggesting that HbXIP2;1 could take part in a wide range of cellular processes. This is particularly relevant to the rubber-producing laticiferous system, where this isoform was found to be up-regulated during tapping and ethylene treatments. Furthermore, the XIP transcriptional pattern is significantly correlated to latex production level. Structural comparison with SoPIP2;1 from Spinacia oleracea species provides new insights into the possible role of structural checkpoints by which HbXIP2;1 ensures glycerol transfer across the membrane. From these results, we discuss the physiological involvement of glycerol and HbXIP2;1 in water homeostasis and carbon stream of challenged laticifers. The characterization of HbXIP2;1 during rubber tree tapping lends new insights into molecular and physiological response processes of laticifer metabolism in the context of latex exploitation.

  7. Bi-functionality of Opisthorchis viverrini aquaporins.

    Science.gov (United States)

    Geadkaew, Amornrat; von Bülow, Julia; Beitz, Eric; Tesana, Smarn; Vichasri Grams, Suksiri; Grams, Rudi

    2015-01-01

    Aquaporins (AQP) are essential mediators of water regulation in all living organisms and members of the major intrinsic protein (MIP) superfamily of integral membrane proteins. They are potential vehicles or targets for chemotherapy, e.g. in Trypanosoma brucei melarsoprol and pentamidine uptake is facilitated by TbAQP-2. Transcriptome data suggests that there are at least three active aquaporins in the human liver fluke, Opisthorchis viverrini, OvAQP-1, 2 and 3, and crude RNA silencing of OvAQP-1 and 2 has recently been shown to affect parasite swelling in destilled water. In the present work we demonstrate that OvAQP-3 is a major water-conducting channel of the parasite, that it can be detected from the newly excysted juvenile to the adult stage and that it is present in major tissues of the parasite. Furthermore, a comparative functional characterization of the three parasite AQPs was performed by using Xenopus oocyte swelling and yeast phenotypic assays. OvAQP-1, OvAQP-2, and OvAQP-3 were found to conduct water and glycerol while only the latter two were also able to conduct urea. In addition, all OvAQPs were found to transport ammonia and methylamine. Our findings demonstrate that the sequence-based classification into orthodox aquaporins and glycerol-conducting aquaglyceroporins is not functionally conserved in the parasite and implicate a broder range of functions for these channels.

  8. Cerebrospinal fluid aquaporin-4-immunoglobulin G disrupts blood brain barrier

    DEFF Research Database (Denmark)

    Asgari, Nasrin; Berg, Carsten Tue; Mørch, Marlene Thorsen;

    2015-01-01

    To clarify the significance of immunoglobulin G autoantibody specific for the astrocyte water channel aquaporin-4 in cerebrospinal fluid, aquaporin-4-immunoglobulin G from a neuromyelitis optica patient was administered intrathecally to naïve mice, and the distribution and pathogenic impact...

  9. The H{sub 1}–H{sub 2} domain of the α{sub 1} isoform of Na{sup +}–K{sup +}–ATPase is involved in ouabain toxicity in rat ventricular myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Chen; Li, Jun-xia; Guo, Hui-cai; Zhang, Li-nan; Guo, Wei; Meng, Jing; Wang, Yong-li, E-mail: wangyongli@gmail.com

    2012-07-01

    The composition of different isoforms of Na{sup +}-K{sup +}-ATPase (NKA, Na/K pump) in ventricular myocytes is an important factor in determining the therapeutic effect and toxicity of cardiac glycosides (CGs) on heart failure. The mechanism whereby CGs cause these effects is still not completely clear. In the present study, we prepared two site-specific antibodies (SSA78 and WJS) against the H{sub 1}–H{sub 2} domain of α{sub 1} and α{sub 2} isoforms of NKA in rat heart, respectively, and compared their influences on the effect of ouabain (OUA) in isolated rat ventricular myocytes. SSA78 or WJS, which can specifically bind with the α{sub 1} or α{sub 2} isoform, were assessed with enzyme linked immunosorbent assay (ELISA), Western blot and immunofluorescent staining methods. Preincubation of myocytes with SSA78 inhibited low OUA affinity pump current but not high OUA affinity pump current, reduced the rise in cytosolic calcium concentration ([Ca{sup 2+}]{sub i}), attenuated mitochondrial Ca{sup 2+} overload, restored mitochondrial membrane potential reduction, and delayed the decrease of the myocardial contractile force as well as the occurrence of arrhythmic contraction induced by high concentrations (1 mM) but not low concentrations (1 μM) of OUA. Similarly, preincubation of myocytes with WJS inhibited high OUA affinity pump current, reduced the increase of [Ca{sup 2+}]{sub i} and the contractility induced by 1 μM but not that induced by 1 mM OUA. These results indicate that the H{sub 1}–H{sub 2} domain of the NKA α{sub 1} isoform mediates OUA-induced cardiac toxicity in rat ventricular myocytes, and inhibitors for this binding site may be used as an adjunct to CGs treatment for cardiovascular disease. -- Highlights: ► We prepared two antibodies against the H{sub 1}-H{sub 2} domain of α{sub 1} and α{sub 2} isoforms of NKA. ► The H{sub 1}-H{sub 2} domain of the NKA α{sub 1} isoform mediates OUA-induced cardiac toxicity. ► The H{sub 1}-H{sub 2

  10. Aquaporins: highly regulated channels controlling plant water relations.

    Science.gov (United States)

    Chaumont, François; Tyerman, Stephen D

    2014-04-01

    Plant growth and development are dependent on tight regulation of water movement. Water diffusion across cell membranes is facilitated by aquaporins that provide plants with the means to rapidly and reversibly modify water permeability. This is done by changing aquaporin density and activity in the membrane, including posttranslational modifications and protein interaction that act on their trafficking and gating. At the whole organ level aquaporins modify water conductance and gradients at key "gatekeeper" cell layers that impact on whole plant water flow and plant water potential. In this way they may act in concert with stomatal regulation to determine the degree of isohydry/anisohydry. Molecular, physiological, and biophysical approaches have demonstrated that variations in root and leaf hydraulic conductivity can be accounted for by aquaporins but this must be integrated with anatomical considerations. This Update integrates these data and emphasizes the central role played by aquaporins in regulating plant water relations.

  11. Isolation and expression of an aquaporin-like gene VfPIP1 in Vicia faba

    Institute of Scientific and Technical Information of China (English)

    CUI Xianghuan; HAO Fushun; CHEN Hui; CAI Jinghui; CHEN Jia; WANG Xuechen

    2005-01-01

    To explore the effects of aquaporins on stomatal movement, we isolated a full length cDNA of aquaporin-like gene VfPIP1 ( Vicia faba plasma membrane intrinsic protein gene, GenBank accession number: AY667436), which encodes for a 290-amino-acid polypeptide, from Vicia faba leaf epidermis by 5′/3′ RACE (Rapid Amplification of cDNA Ends). The analyses of VfPIP1 transmembrane regions and amino acid sequence show that VfPIP1 owns six membrane-spanning domains and the special plasma membrane signature sequences GGGANXXXXGY and TGI/TNPARSL/FGAAI/VI/VF/YN, and it should be a member of PIP1 subfamily. The results of in situ hybridization and Northern blot indicate that VfPIP1 is strongly expressed in guard cells and induced by ABA. Hereby, VfPIP1 may be involved in the water-transmembrane movement of guard cells.

  12. Expression of aquaporin-1 in SMMC-7221 liver carcinoma cells promotes cell migration

    Institute of Scientific and Technical Information of China (English)

    LI Yongming; FENG Xuechao; YANG Hong; MA Tonghui

    2006-01-01

    Migration of tumor cells is a crucial step in tumor invasion and metastasis. Here we provide evidence that aquaporin expression is involved in tumor cell migration. RT-PCR, immunofluorescence and Western blot analysis demonstrated the AQP1 protein expression on the plasma membrane of SMMC-7221 human hepatoma cells. SMMC-7221 cell clones with high (SMMC-7221hPf) and low (SMMC-7221/Pf) water permeability were identified by functional assays with corresponding high and low AQP1 expression. Cell migration rate was remarkably higher in SMMC-7221hPf cells than SMMC-7221/Pf cells, assessed by Boyden chamber and wound healing assays, whereas cell growth and adhesion were not different. Adenovirus-mediated AQP1 expression in SMMC-7221/Pf cells increased their water permeability and migration rate. These results provide the first evidence that aquaporin-mediated membrane water permeability enhances tumor cell migration and may be associated with tumor invasion and metastasis.

  13. ApoE isoform-dependent changes in hippocampal synaptic function

    Directory of Open Access Journals (Sweden)

    Sullivan Patrick M

    2009-05-01

    Full Text Available Abstract The lipoprotein receptor system in the hippocampus is intimately involved in the modulation of synaptic transmission and plasticity. The association of specific apoE isoform expression with human neurodegenerative disorders has focused attention on the role of these apoE isoforms in lipoprotein receptor-dependent synaptic modulation. In the present study, we used the apoE2, apoE3 and apoE4 targeted replacement (TR mice along with recombinant human apoE isoforms to determine the role of apoE isoforms in hippocampus area CA1 synaptic function. While synaptic transmission is unaffected by apoE isoform, long-term potentiation (LTP is significantly enhanced in apoE4 TR mice versus apoE2 TR mice. ApoE isoform-dependent differences in LTP induction require NMDA-receptor function, and apoE isoform expression alters activation of both ERK and JNK signal transduction. Acute application of specific apoE isoforms also alters LTP induction while decreasing NMDA-receptor mediated field potentials. Furthermore, acute apoE isoform application does not have the same effects on ERK and JNK activation. These findings demonstrate specific, isoform-dependent effects of human apoE isoforms on adult hippocampus synaptic plasticity and highlight mechanistic differences between chronic apoE isoform expression and acute apoE isoform exposure.

  14. Chronic noradrenaline increases renal expression of NHE-3, NBC-1, BSC-1 and aquaporin-2.

    Science.gov (United States)

    Sonalker, Prajakta A; Tofovic, Stevan P; Bastacky, Sheldon I; Jackson, Edwin K

    2008-05-01

    1. Because chronic activation of the renal sympathetic nervous system promotes sodium and water retention, it is conceivable that long-term exposure of the kidney to the sympathetic neurotransmitter noradrenaline upregulates the expression of key renal epithelial transport systems. 2. To test this hypothesis, we used immunoblotting of renal cortical and medullary tissue to investigate the abundance of major transport systems expressed along the renal tubule in response to long-term (15 days) infusions of noradrenaline (600 ng/min) in rats. 3. Mean arterial blood pressure and heart rate were significantly elevated in rats receiving chronic infusions of noradrenaline (128 +/- 10 mmHg and 492 +/- 16 b.p.m., respectively) compared with animals treated with saline only (89 +/- 3 mmHg and 376 +/- 14 b.p.m., respectively). 4. Chronic infusions of noradrenaline also increased the protein abundance of the cortical Na(+)/H(+) exchanger isoform 3 (NHE-3; 2.5-fold; P = 0.0142), the cortical sodium-bicarbonate cotransporter NBC-1 (2.5-fold; P = 0.0067), the bumetanide-sensitive sodium-potassium-chloride cotransporter BSC-1/NKCC2 in the inner stripe of outer medulla (threefold; P = 0.0020) and aquaporin-2 in the inner medulla (twofold; P = 0.0039). 5. In contrast, noradrenaline did not significantly affect expression of the thiazide-sensitive Na(+)-Cl(-) cotransporter in the cortex, Na(+)/K(+)-ATPase-alpha(1) in the cortex and inner stripe of the outer or inner medulla, the inwardly rectifying K(+) channel (ROMK-1) in the inner stripe of the outer medulla or aquaporin-1 in the cortex or inner medulla. Noradrenaline did significantly, but modestly (less than twofold), increase aquaporin-1 in the inner stripe of the outer medulla. 6. We conclude that noradrenaline-induced increases in the expression of NHE-3, NBC-1, BSC-1 and aquaporin-2 are likely to play an important role in the regulation of salt and water transport by noradrenaline in the kidney and may explain, at least in

  15. Biomimetic aquaporin membranes coming of age

    DEFF Research Database (Denmark)

    Tang, Chuyang; Wang, Zhining; Petrinić, Irena

    2015-01-01

    Membrane processes have been widely used for water purification because of their high stability, efficiency, low energy requirement and ease of operation. Traditional desalting membranes are mostly dense polymeric films with a "trade off" effect between permeability and selectivity. Biological...... membranes, on the other hand, can perform transport in some cases with exceptional flux and rejection properties. In particular the discovery of selective water channel proteins - aquaporins - has prompted interest in using these proteins as building blocks for new types of membranes. The major challenge...

  16. Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends

    Science.gov (United States)

    Bernardino, Raquel L.; Marinelli, Raul A.; Maggio, Anna; Gena, Patrizia; Cataldo, Ilaria; Alves, Marco G.; Svelto, Maria; Oliveira, Pedro F.; Calamita, Giuseppe

    2016-01-01

    Aquaporins (AQPs) are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs). Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field. PMID:27409609

  17. Molecular dynamics insights into human aquaporin 2 water channel.

    Science.gov (United States)

    Binesh, A R; Kamali, R

    2015-12-01

    In this study, the first molecular dynamics simulation of the human aquaporin 2 is performed and for a better understanding of the aquaporin 2 permeability performance, the characteristics of water transport in this protein channel and key biophysical parameters of AQP2 tetramer including osmotic and diffusive permeability constants and the pore radius are investigated. For this purpose, recently recovered high resolution X-ray crystal structure of` the human aquaporin 2 is used to perform twenty nanosecond molecular dynamics simulation of fully hydrated tetramer of this protein embedded in a lipid bilayer. The resulting water permeability characteristics of this protein channel showed that the water permeability of the human AQP2 is in a mean range in comparison with other human aquaporins family. Finally, the results reported in this research demonstrate that molecular dynamics simulation of human AQP2 provided useful insights into the mechanisms of water permeation and urine concentration in the human kidney.

  18. Tonoplast aquaporins facilitate lateral root emergence

    DEFF Research Database (Denmark)

    Reinhardt, Hagen; Hachez, Charles; Bienert, Manuela Désirée

    2016-01-01

    that, in Arabidopsis (Arabidopsis thaliana), the highly abundant tonoplast AQP isoforms AtTIP1;1, AtTIP1;2, and AtTIP2;1 facilitate the emergence of new lateral root primordia (LRPs). The number of lateral roots was strongly reduced in the triple tip mutant, whereas the single, double, and triple tip...... could be fully rescued by expressing AtTIP2;1 under its native promoter. We conclude that TIP isoforms allow the spatial and temporal fine-tuning of cellular water transport, which is critically required during the highly regulated process of LRP morphogenesis and emergence....

  19. Aquaporin-1: new developments and perspectives for peritoneal dialysis.

    Science.gov (United States)

    Devuyst, Olivier; Yool, Andrea J

    2010-01-01

    Peritoneal dialysis involves diffusive and convective transport and osmosis through the highly vascularized peritoneal membrane. Several lines of evidence have demonstrated that the water channel aquaporin-1 (AQP1) corresponds to the ultrasmall pore predicted by the model of peritoneal transport. Proof-of-principle studies have shown that upregulation of the expression of AQP1 in peritoneal capillaries results in increased water permeability and ultrafiltration, without affecting the osmotic gradient or small solute permeability. Conversely, studies in Aqp1 mice have shown that haplo-insufficiency for AQP1 results in significant attenuation of water transport. Recent studies have demonstrated that AQP1 is involved in the migration of different cell types, including endothelial cells. In parallel, chemical screening has identified lead compounds that could act as antagonists or agonists of AQPs, with description of putative binding sites and potential mechanisms of gating the water channel. By modulating water transport, these pharmacological agents could have clinically relevant effects in targeting specific tissues or disease states.

  20. Anti-aquaporin-4 antibody-positive dorsal midbrain syndrome.

    Science.gov (United States)

    Lee, Juyoun; Jeong, Seong-Hae; Park, Sang Min; Sohn, Eun Hee; Lee, Ae Young; Kim, Jae-Moon; Jo, Hyun-Jin; Lee, Yeon-Hee; Kim, Ji-Soo

    2015-04-01

    Neuromyelitis optica spectrum disorders (NMOSD) can cause various ocular motor disorders in addition to optic neuritis. Ocular motor findings associated with NMOSD include spontaneous vertical and gaze-evoked nystagmus, wall-eyed bilateral internuclear ophthalmoplegia, and trochlear nerve palsy. The association between dorsal midbrain syndrome and anti-aquaporin-4 antibody seropositivity has not been reported. Here, we report a patient displaying typical dorsal midbrain syndrome and anti-aquaporin-4 antibody seropositivity.

  1. Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes.

    Science.gov (United States)

    Bienert, Gerd P; Møller, Anders L B; Kristiansen, Kim A; Schulz, Alexander; Møller, Ian M; Schjoerring, Jan K; Jahn, Thomas P

    2007-01-12

    The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.

  2. Aquaporins in development – a review

    Directory of Open Access Journals (Sweden)

    Wintour E Marelyn

    2005-05-01

    Full Text Available Abstract Water homeostasis during fetal development is of crucial physiologic importance. It depends upon maternal fetal fluid exchange at the placenta and fetal membranes, and some exchange between fetus and amniotic fluid can occur across the skin before full keratinization. Lungs only grow and develop normally with fluid secretion, and there is evidence that cerebral spinal fluid formation is important in normal brain development. The aquaporins are a growing family of molecular water channels, the ontogeny of which is starting to be explored. One question that is of particular importance is how well does the rodent (mouse, rat fetus serve as a model for long-gestation mammals such as sheep and human? This is particularly important for organs such as the lung and the kidney, whose development before birth is very much less in rodents than in the long-gestation species.

  3. Ammonia induces upregulation of aquaporin-4 in neocortical astrocytes of rats through the p38 mitogen-activated protein kinase pathway

    Institute of Scientific and Technical Information of China (English)

    PAN Cai-fei; ZHU Sheng-mei; ZHENG Yue-ying

    2010-01-01

    Background Astrocyte swelling is an important consequence of hepatic encephalopathy, and aquaporin-4 has been reported to play a vital role in this swelling. Ammonia causes astrocyte swelling and is also known to modulate aquaporin-4 expression in the astrocyte foot processes. The purpose of this study was to explore the mechanism of ammonia-induced aquaporin-4 expression, which has been suggested to involve the p38 mitogen-activated protein kinase pathway.Methods We exposed cultured astrocytes to ammonium chloride, an in vitro model of hepatic encephalopathy. The purity of cultured astrocytes was evaluated by fluorescent glial fibrillary acidic protein labeling; cell morphology was assessed by light microscopy; the expression of aquaporin-4, phospho-p38, and p38 were detected by Western blotting analysis. Statistical analysis was performed by one-way factorial analysis of variance, and the relationship between variables was calculated by linear regression using SPSS version 13.0 program for Windows (SPSS, Chicago, IL, USA).Results The purity of cultured astrocytes was (96.6 ±1.4)%. Astrocytes swelled significantly when exposed to 5 mmol/L ammonium chloride for 24 hours as compared to non-exposed astrocytes. Co-treatment with 10 μmol/L SB203580 (an inhibitor of p38) attenuated the degree of ammonium chloride induced astrocyte swelling. Western blotting analysis revealed that the expression levels of phospho-p38 and aquaporin-4 in ammonium chloride treated cells were significantly increased relative to the control group (P <0.001); SB203580 co-treatment inhibited the increased expression of phospho-p38 and aquaporin-4 relative to the ammonium chloride treated group (P=0.002 and P=0.015 respectively). The phosphorylation of p38 and upregulation of aquaporin-4 were highly correlated (r=0.909). There were no significant differences in total p38 expression among the groups (P=0.341).Conclusions Ammonium chloride induced upregulation of aquaporin-4 in astrocytes is

  4. Metal ion toxins and brain aquaporin-4 expression: an overview

    Directory of Open Access Journals (Sweden)

    Adriana eXimenes-Da-Silva

    2016-06-01

    Full Text Available Metal ions such as iron, zinc, and manganese are essential to metabolic functions, protein synthesis, neurotransmission, and antioxidant neuroprotective mechanisms. Conversely, non-essential metals such as mercury and lead are sources of human intoxication due to occupational activities or environmental contamination. Essential or non-essential metal accumulation in the central nervous system (CNS results in changes in blood-brain barrier (BBB permeability, as well as triggering microglia activation and astrocyte reactivity and changing water transport through the cells, which could result in brain swelling. Aquaporin-4 is the main water channel in the CNS, is expressed in astrocyte foot processes in brain capillaries and along the circumventricular epithelium in the ventricles, and has important physiological functions in maintaining brain osmotic homeostasis and supporting brain excitability through regulation of the extracellular space. Some evidence has pointed to a role of AQP4 during metal intoxication in the brain, where it may act in a dual form as a neuroprotector or a mediator of the development of oxidative stress in neurons and astrocytes, resulting in brain swelling and neuronal damage. This mini-review presents the way some metal ions affect changes in AQP4 expression in the CNS and discuss the ways in which water transport in brain cells can be involved in brain damage.

  5. Nuclear Receptor Regulation of Aquaporin-2 in the Kidney

    Directory of Open Access Journals (Sweden)

    Xiao-Yan Zhang

    2016-07-01

    Full Text Available Aquaporin-2 (AQP2 is a vasopressin-regulated water channel responsible for regulating water reabsorption through the apical plasma membrane of the principal cells of renal collecting ducts. It has been found that dysregulation and dysfunction of AQP2 cause many disorders related to water balance in people and animals, including polyuria and dilutional hyponatremia. Classically, AQP2 mRNA and protein expression and its membrane translocation are regulated by systemic vasopressin involving short-term regulation of AQP2 trafficking to and from the apical plasma membrane and long-term regulation of the total amount of the AQP2 protein in the cell. Recently, increasing evidence has demonstrated that collecting duct AQP2 expression and membrane translocation are also under the control of many other local factors, especially nuclear receptors. Here, we briefly review the progress of studies in this area and discuss the role of nuclear receptors in the regulation of water reabsorption via affecting AQP2 expression and function.

  6. Aquaporin-1 Expression in Proliferative Vitreoretinopathy and in Epiretinal Membranes

    Directory of Open Access Journals (Sweden)

    Elie Motulsky

    2014-01-01

    Full Text Available Purpose. Aquaporin-1 (AQP1 is involved in cell migration and proliferation; therefore, the purpose of the study was to investigate its expression in proliferative vitreoretinopathy (PVR and epiretinal membranes (ERM. Methods. 19 membranes from PVR and ERM were collected following eye surgery. AQP1 mRNA and protein expressions were determined by RT-qPCR and immunofluorescence in the membranes from PVR and ERM. Results. AQP1 mRNA and protein were expressed in both PVR and ERM as shown by RT-qPCR and immunofluorescence. AQP1 protein expression was heterogeneous among and between PVR and ERM and colocalized with alpha-smooth muscle actin (αSMA and with glial fibrillary acidic protein (GFAP. There were a higher percentage of cells coexpressing AQP1 and αSMA than AQP1 and GFAP. GFAP and αSMA did not colocalize. Conclusion. Our data show for the first time AQP1 expression in both PVR and ERM. AQP1 is expressed mostly by the αSMA-positive cells, presumably myofibroblasts, but also by GFAP-positive cells, assumed to be glial cells. These original findings warrant further functional investigations aiming at studying the potential role of AQP1 in cell migration and proliferation occurring during the development of PVR and ERM.

  7. Maize black Mexican sweet suspension cultured cells are a convenient tool for studying aquaporin activity and regulation.

    Science.gov (United States)

    Cavez, Damien; Hachez, Charles; Chaumont, François

    2009-09-01

    Aquaporins (AQPs) are channel proteins that facilitate and regulate the permeation of water across biological membranes. Black Mexican sweet suspension cultured cells are a convenient model for studying the regulation of maize AQP expression and activity. Among other advantages, a single cell system allows the contribution of plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) to the membrane water permeability coefficient (P(f)) to be determined using biophysical measurement methods, such as the cell pressure probe or protoplast swelling assay. We generated a transgenic cell culture line expressing a tagged version of ZmPIP2;6 and used this material to demonstrate that the ZmPIP2;6 and ZmPIP2;1 isoforms physically interact. This kind of interaction could be an additional mechanism for regulating membrane water permeability by acting on the activity and/or trafficking of PIP hetero-oligomers.

  8. Histamine H3-receptor isoforms.

    Science.gov (United States)

    Bakker, R A

    2004-10-01

    Increasing evidence supports a role for HA as a neurotransmitter and neuromodulator in various brain functions, including emotion, cognition, and feeding. The recent cloning of the histamine H3 receptor allowed for the subsequent cloning of a variety of H3 receptor isoforms from different species as well as the H4 receptor. As a result a wide variety of H3-receptor isoforms are now known that display differential brain expression patterns and signalling properties. These recent discoveries are discussed in view of the growing interest of the H3 receptor as a target for the development of potential therapeutics.

  9. Aquaporin-1 and Aquaporin-3 Expressions in the Intervertebral Disc of Rats with Aging

    Directory of Open Access Journals (Sweden)

    Mustafa Sarsılmaz

    2012-12-01

    Full Text Available Objective: The intervertebral disc (IVD undergoes biochemical and morphologic degenerative changes during the process of aging. Aquaporins (AQPs are a family of water channel proteins that facilitate water and small solute movement in tissues and may have a potential role in the aging degeneration of IVDs. One of the important problems in understanding disc degeneration is to find cellular molecules which contribute to the pathogenesis of IVDs. XThe aim of this study was to demonstrate the expression of aquaporin 1 and 3 in nucleus pulposus (NP, annulus fibrosus (AF cells of rat lumbar intervertebral discs from both young and aged animals using immunohistochemistry.Material and Methods: Twenty Wistar-albino rats were included in the study. The rats were separated into two groups: 2-month-old rats (n=10 as the young group, 18-month-old rats (n=10 as the old group. The intervertebral disc tissues obtained from the lumbar spine (L1–L4, 4 discs were used for immunohistochemical staining of AQP-1 and 3. Results: This study demonstrated that AQP-1 and AQP-3 immunoreactivity significantly decreased in NP and AF of aged rats compared to the young rats. Conclusion: We suggest that AQP-1 and 3 may contribute to the age related degeneration of the intervertebral disc.

  10. Aquaporins in the wild: natural genetic diversity and selective pressure in the PIP gene family in five Neotropical tree species

    Directory of Open Access Journals (Sweden)

    Vendramin Giovanni G

    2010-06-01

    Full Text Available Abstract Background Tropical trees undergo severe stress through seasonal drought and flooding, and the ability of these species to respond may be a major factor in their survival in tropical ecosystems, particularly in relation to global climate change. Aquaporins are involved in the regulation of water flow and have been shown to be involved in drought response; they may therefore play a major adaptive role in these species. We describe genetic diversity in the PIP sub-family of the widespread gene family of Aquaporins in five Neotropical tree species covering four botanical families. Results PIP Aquaporin subfamily genes were isolated, and their DNA sequence polymorphisms characterised in natural populations. Sequence data were analysed with statistical tests of standard neutral equilibrium and demographic scenarios simulated to compare with the observed results. Chloroplast SSRs were also used to test demographic transitions. Most gene fragments are highly polymorphic and display signatures of balancing selection or bottlenecks; chloroplast SSR markers have significant statistics that do not conform to expectations for population bottlenecks. Although not incompatible with a purely demographic scenario, the combination of all tests tends to favour a selective interpretation of extant gene diversity. Conclusions Tropical tree PIP genes may generally undergo balancing selection, which may maintain high levels of genetic diversity at these loci. Genetic variation at PIP genes may represent a response to variable environmental conditions.

  11. Aquaporins: Their role in cholestatic liver disease

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    This review focuses on currant knowledge on hepato-cyte aquaporins (AQPs) and their significance in bile formation and cholestasis. Canalicular bile secretion results from a combined interaction of several solute transporters and AQP water channels that facilitate wa-ter flow in response to the osmotic gradients created. During choleresis, hepatocytes rapidly increase their canalicular membrane water permeability by modulat-ing the abundance of AQP8. The question was raised as to whether the opposite process, i.e. a decreased canalicular AQP8 expression would contribute to the development of cholestasis. Studies in several experi-mental models of cholestasis, such as extrahepatic obstructive cholestasis, estrogen-induced cholestasis, and sepsis-induced cholestasis demonstrated that the protein expression of hepatocyte AQP8 was impaired. In addition, biophysical studies in canalicular plasma membranes revealed decreased water permeability as-sociated with AQP8 protein downregulation. The com-bined alteration in hepatocyte solute transporters and AQP8 would hamper the efficient coupling of osmotic gradients and canalicular water flow. Thus cholestasis may result from a mutual occurrence of impaired sol-ute transport and decreased water permeability.

  12. Aquaporin-4 and traumatic brain edema

    Institute of Scientific and Technical Information of China (English)

    XU Miao; SU Wei; XU Qiu-ping

    2010-01-01

    Brain edema leading to an expansion of brain volume has a crucial impact on morbidity and mortal-ity following traumatic brain injury as it increases intracra-nial pressure, impairs cerebral perfusion and oxygenation,and contributes to additional ischemic injuries.Classically,two major types of traumatic brain edema exist: "vasogenic"and "cytotoxic/cellular".However, the cellular and molecu-lar mechanisms contributing to the development/resolution of traumatic brain edema are poorly understood and no ef-fective drugs can be used now.Aquaporin-4 (AQP4) is a water-channel protein expressed strongly in the brain, pre-dominantly in astrocyte foot processes at the borders be-tween the brain parenchyma and major fluid compartments, including cerebrospinal fluid and blood.This distribution suggests that AQP4 controls water fluxes into and out of the brain parenchyma.In cytotoxic edema, AQP4 deletion slows the rate of water entry into brain, whereas in vasogenic edema, AQP4 deletion reduces the rate of water outflow from brain parenchyma.AQP4 has been proposed as a novel drug target in brain edema.These findings sug-gest that modulation of AQP4 expression or function may be beneficial in traumatic brain edema.

  13. Altered aquaporin expression in glaucoma eyes

    DEFF Research Database (Denmark)

    Tran, Thuy Linh; Bek, Toke; la Cour, Morten

    2014-01-01

    Aquaporins (AQP) are channels in the cell membrane that mainly facilitate a passive transport of water. In the eye, AQPs are expressed in the ciliary body and retina and may contribute to the pathogenesis of glaucoma and optic neuropathy. We investigated the expression of AQP1, AQP3, AQP4, AQP5......, AQP7 and AQP9 in human glaucoma eyes compared with normal eyes. Nine glaucoma eyes were examined. Of these, three eyes were diagnosed with primary open angle glaucoma; three eyes had neovascular glaucoma; and three eyes had chronic angle-closure glaucoma. Six eyes with normal intraocular pressure...... of AQP7 and AQP9 in the nonpigmented ciliary epithelium and the staining intensities were significantly decreased in glaucoma eyes (p = 0.003; p = 0.018). AQP7 expression in the Müller cell endfeet was increased (p = 0.046), and AQP9 labelling of the retinal ganglion cells (RGC) showed decreased...

  14. Aquaporin-2 water channels in spontaneously hypertensive rats.

    Science.gov (United States)

    Buemi, Michele; Nostro, Lorena; Di Pasquale, Giuseppe; Cavallaro, Emanuela; Sturiale, Alessio; Floccari, Fulvio; Aloisi, Carmela; Ruello, Antonella; Calapai, Gioacchino; Corica, Francesco; Frisina, Nicola

    2004-12-01

    Vasopressin (AVP), an antidiuretic hormone, is known to induce hypervolemia and to regulate the renal expression of aquaporin-2 (AQP2) water channels, but it is not yet known whether the latter are involved in the pathogenesis of essential hypertension. The aim of the present study was therefore to make a comparative study of blood pressure (BP), urinary volume (UV), urinary osmolarity (uOsm), urinary AQP2 (uAQP2), and plasma AVP levels (PAVP) in male spontaneously hypertensive rats (SHR; n = 30) at 3, 7, and 12 weeks of age and in male Wistar-Kyoto rats (WKY, n = 30), also after the subcutaneous administration of OPC-31260 (OPC), a human AVP V(2) receptor antagonist. At 3 weeks, SHR had markedly higher uOsm and lower UV levels than WKY. At 7 weeks, SHR were hypertensive, showing increased uAQP2, PAVP, and uOsm levels and a decreased UV. At 12 weeks, no significant changes were observed in this condition. At 7 and 12 weeks of age, OPC-treated WKY rats showed significant reduction in BP and uOsm and increase in UV with respect to untreated animals. From 3 weeks of age, OPC-treated SHR presented significantly lower BP levels, higher UV levels, and lower uOsm than untreated animals. In treated WKY and SHR, uAQP2 levels were lower than in untreated animals. The PAVP appeared to be higher in OPC-treated rats from both strains. These findings suggest that AVP and the AQP2 are involved in the pathogenesis of hypertension in SHR.

  15. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC.

    Science.gov (United States)

    Schwab, Ryan S; Ihnatovych, Ivanna; Yunus, Sharifah Z S A; Domaradzki, Tera; Hofmann, Wilma A

    2013-05-01

    Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms.

  16. Loop A Is Critical for the Functional Interaction of Two Beta vulgaris PIP Aquaporins

    Science.gov (United States)

    Jozefkowicz, Cintia; Rosi, Pablo; Sigaut, Lorena; Soto, Gabriela; Pietrasanta, Lía Isabel; Amodeo, Gabriela; Alleva, Karina

    2013-01-01

    Research done in the last years strongly support the hypothesis that PIP aquaporin can form heterooligomeric assemblies, specially combining PIP2 monomers with PIP1 monomers. Nevertheless, the structural elements involved in the ruling of homo versus heterooligomeric organization are not completely elucidated. In this work we unveil some features of monomer-monomer interaction in Beta vulgaris PIP aquaporins. Our results show that while BvPIP2;2 is able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. The lack of functional interaction between BvPIP2;1 and BvPIP1;1 was further corroborated by dose-response curves of water permeability due to aquaporin activity exposed to different acidic conditions. We also found that BvPIP2;1 is unable to translocate BvPIP1;1-ECFP from an intracellular position to the plasma membrane when co-expressed, as BvPIP2;2 does. Moreover we postulate that the first extracellular loop (loop A) of BvPIP2;1, could be relevant for the functional interaction with BvPIP1;1. Thus, we investigate BvPIP2;1 loop A at an atomic level by Molecular Dynamics Simulation (MDS) and by direct mutagenesis. We found that, within the tetramer, each loop A presents a dissimilar behavior. Besides, BvPIP2;1 loop A mutants restore functional interaction with BvPIP1;1. This work is a contribution to unravel how PIP2 and PIP1 interact to form functional heterooligomeric assemblies. We postulate that BvPIP2;1 loop A is relevant for the lack of functional interaction with BvPIP1;1 and that the monomer composition of PIP assemblies determines their functional properties. PMID:23483963

  17. Aquaporins in Coffea arabica L.: Identification, expression, and impacts on plant water relations and hydraulics.

    Science.gov (United States)

    Miniussi, Matilda; Del Terra, Lorenzo; Savi, Tadeja; Pallavicini, Alberto; Nardini, Andrea

    2015-10-01

    Plant aquaporins (AQPs) are involved in the transport of water and other small solutes across cell membranes, and thus play major roles in the regulation of plant water balance, as well as in growth regulation and response to abiotic stress factors. Limited information is currently available about the presence and role of AQPs in Coffea arabica L., despite the economic importance of the species and its vulnerability to drought stress. We identified candidate AQP genes by screening a proprietary C. arabica transcriptome database, resulting in the identification of nine putative aquaporins. A phylogenetic analysis based on previously characterized AQPs from Arabidopsis thaliana and Solanum tuberosum allowed to assign the putative coffee AQP sequences to the Tonoplast (TIP) and Plasma membrane (PIP) subfamilies. The possible functional role of coffee AQPs was explored by measuring hydraulic conductance and aquaporin gene expression on leaf and root tissues of two-year-old plants (C. arabica cv. Pacamara) subjected to different experimental conditions. In a first experiment, we tested plants for root and leaf hydraulic conductance both before dawn and at mid-day, to check the eventual impact of light on AQP activity and plant hydraulics. In a second experiment, we measured plant hydraulic responses to different water stress levels as eventually affected by changes in AQPs expression levels. Our results shed light on the possible roles of AQPs in the regulation of C. arabica hydraulics and water balance, opening promising research lines to improve the sustainability of coffee cultivation under global climate change scenarios.

  18. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC

    Energy Technology Data Exchange (ETDEWEB)

    Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.; Domaradzki, Tera [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States); Hofmann, Wilma A., E-mail: whofmann@buffalo.edu [Department of Physiology and Biophysics, University at Buffalo—State University of New York, Buffalo, NY (United States)

    2013-05-01

    Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms. - Highlights: ► Two NoLS have been identified in the myosin IC isoform B sequence. ► Both NoLS are necessary for myosin IC isoform B specific nucleolar localization. ► First mechanistic explanation of functional differences between the isoforms.

  19. Aquaporin 1 Facilitated Hepatocellular Carcinoma SMMC7221 Cell Migration Associated with Water Permeability

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ai-li; LI Jiang; WANG Yan-qing; ZAKNROU Zohra; MA Tong-hui; LI Xiao-meng

    2011-01-01

    The authors investigated the regulation of human aquaporin l(hAQPl) and the involvement of aquaporin l(AQPl) in the migration of human hepatocellular carcinoma SMMC-7221 cells using RNA intereference technology.Firstly, two short hairpin RNA(shRNA) constructs in PBSU6 vector were reconstructed and their knockdown effects were identified in SMMC-7221 cells. Next, the involvement of endogenous hAQPl in regulating the migration of SMMC-7221 cells was investigated via siRNA technology. HAQPl-shRNA can specifically inhibit AQPl dependent osmotic water permeability. Meanwhile the migration of SMMC-7221 cells was inhibited remarkably after silencing AQPl by performing transwell cell migration assay and in vitro wound healing assay. Furthermore, in the presence of an inhibitor HgCl2, the water permeability of the cell membrane was remarkably decreased, the expression of AQPl was upregulated after HgCl2 treatment and the cell movement was decreased at the moment. Increased AQPl cannot attenuate cell migration ability when cell membrane loses its water permeability function. This demonstrates that the cell migration was remarkably related to the transporting water function of cell membrane.

  20. Detection of anti-aquaporin-4 autoantibodies in the sera of Chinese neuromyelitis optica patients

    Institute of Scientific and Technical Information of China (English)

    Miao Li; Weiheng Su; Jie Wang; Francesco Pisani; Antonio Frigeri; Tonghui Ma

    2013-01-01

    In this study, we recruited 10 neuromyelitis optica patients, two multiple sclerosis patients and two myelitis patients. Chinese hamster lung fibroblast (V79) cells transfected with a human aquaporin-4-mCherry fusion protein gene were used to detect anti-aquaporin-4 antibody in neuromyelitis optica patient sera by immunofluorescence. Anti-aquaporin-4 autoantibody was stably detected by immunofluorescence in neuromyelitis optica patient sera exclusively. The sensitivity of the assay for neuromyelitis optica was 90% and the specificity for neuromyelitis optica was 100%. The anti-aquaporin-4 antibody titers in sera were tested with serial dilutions until the signal disappeared. A positive correlation was detected between Expanded Disability Status Scale scores and serum anti-aquaporin-4 antibody titers. The anti-aquaporin-4 antibody assay is highly sensitive and specific in the sera of Chinese neuromyelitis optica patients. Detection of aquaporin-4 autoantibody is important for the diagnosis and treatment of neuromyelitis optica.

  1. Detection of anti-aquaporin-4 autoantibodies in the sera of Chinese neuromyelitis optica patients.

    Science.gov (United States)

    Li, Miao; Su, Weiheng; Wang, Jie; Pisani, Francesco; Frigeri, Antonio; Ma, Tonghui

    2013-03-15

    In this study, we recruited 10 neuromyelitis optica patients, two multiple sclerosis patients and two myelitis patients. Chinese hamster lung fibroblast (V79) cells transfected with a human aquaporin-4-mCherry fusion protein gene were used to detect anti-aquaporin-4 antibody in neuromyelitis optica patient sera by immunofluorescence. Anti-aquaporin-4 autoantibody was stably detected by immunofluorescence in neuromyelitis optica patient sera exclusively. The sensitivity of the assay for neuromyelitis optica was 90% and the specificity for neuromyelitis optica was 100%. The anti-aquaporin-4 antibody titers in sera were tested with serial dilutions until the signal disappeared. A positive correlation was detected between Expanded Disability Status Scale scores and serum anti-aquaporin-4 antibody titers. The anti-aquaporin-4 antibody assay is highly sensitive and specific in the sera of Chinese neuromyelitis optica patients. Detection of aquaporin-4 autoantibody is important for the diagnosis and treatment of neuromyelitis optica.

  2. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces Cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Helix Nielsen, Claus; Scharff-Poulsen, Peter

    2014-01-01

    cerevisiae was exploited as a host for heterologous expression of human aquaporins. Aquaporin cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human aquaporin was C-terminally tagged with yeast-enhanced GFP to quantify functional expression...... transcription factor and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30oC was due to in vivo mal-folding. Reduction of the expression temperature to 15oC almost completely...... prevented Aquaporin1 mal-folding. Bioimaging of live yeast cells revealed that recombinant Aquaporin-1 accumulated in the yeast plasma membrane. A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation...

  3. Superresolution Imaging of Aquaporin-4 Cluster Size in Antibody-Stained Paraffin Brain Sections.

    Science.gov (United States)

    Smith, Alex J; Verkman, Alan S

    2015-12-15

    The water channel aquaporin-4 (AQP4) forms supramolecular clusters whose size is determined by the ratio of M1- and M23-AQP4 isoforms. In cultured astrocytes, differences in the subcellular localization and macromolecular interactions of small and large AQP4 clusters results in distinct physiological roles for M1- and M23-AQP4. Here, we developed quantitative superresolution optical imaging methodology to measure AQP4 cluster size in antibody-stained paraffin sections of mouse cerebral cortex and spinal cord, human postmortem brain, and glioma biopsy specimens. This methodology was used to demonstrate that large AQP4 clusters are formed in AQP4(-/-) astrocytes transfected with only M23-AQP4, but not in those expressing only M1-AQP4, both in vitro and in vivo. Native AQP4 in mouse cortex, where both isoforms are expressed, was enriched in astrocyte foot-processes adjacent to microcapillaries; clusters in perivascular regions of the cortex were larger than in parenchymal regions, demonstrating size-dependent subcellular segregation of AQP4 clusters. Two-color superresolution imaging demonstrated colocalization of Kir4.1 with AQP4 clusters in perivascular areas but not in parenchyma. Surprisingly, the subcellular distribution of AQP4 clusters was different between gray and white matter astrocytes in spinal cord, demonstrating regional specificity in cluster polarization. Changes in AQP4 subcellular distribution are associated with several neurological diseases and we demonstrate that AQP4 clustering was preserved in a postmortem human cortical brain tissue specimen, but that AQP4 was not substantially clustered in a human glioblastoma specimen despite high-level expression. Our results demonstrate the utility of superresolution optical imaging for measuring the size of AQP4 supramolecular clusters in paraffin sections of brain tissue and support AQP4 cluster size as a primary determinant of its subcellular distribution.

  4. Identification and Expression Analysis of the Barley (Hordeum vulgare L. Aquaporin Gene Family.

    Directory of Open Access Journals (Sweden)

    Runyararo M Hove

    Full Text Available Aquaporins (AQPs are major intrinsic proteins (MIPs that mediate bidirectional flux of water and other substrates across cell membranes, and play critical roles in plant-water relations, dehydration stress responses and crop productivity. However, limited data are available as yet on the contributions of these proteins to the physiology of the major crop barley (Hordeum vulgare. The present work reports the identification and expression analysis of the barley MIP family. A comprehensive search of publicly available leaf mRNA-seq data, draft barley genome data, GenBank transcripts and sixteen new annotations together revealed that the barley MIP family is comprised of at least forty AQPs. Alternative splicing events were likely in two plasma membrane intrinsic protein (PIP AQPs. Analyses of the AQP signature sequences and specificity determining positions indicated a potential of several putative AQP isoforms to transport non-aqua substrates including physiological important substrates, and respond to abiotic stresses. Analysis of our publicly available leaf mRNA-seq data identified notable differential expression of HvPIP1;2 and HvTIP4;1 under salt stress. Analyses of other gene expression resources also confirmed isoform-specific responses in different tissues and/or in response to salinity, as well as some potentially inter-cultivar differences. The work reports systematic and comprehensive analysis of most, if not all, barley AQP genes, their sequences, expression patterns in different tissues, potential transport and stress response functions, and a strong framework for selection and/or development of stress tolerant barley varieties. In addition, the barley data would be highly valuable for genetic studies of the evolutionarily closely related wheat (Triticum aestivum L..

  5. Symbiotic interaction between dinoflagellates and the demosponge Lubomirskia baicalensis: aquaporin-mediated glycerol transport.

    Science.gov (United States)

    Müller, Werner E G; Belikov, Sergey I; Kaluzhnaya, Oxana V; Chernogor, L; Krasko, Anatoli; Schröder, Heinz C

    2009-01-01

    Lake Baikal is rich in endemic sponge species, among them the arborescently growing species Lubomirskia baicalensis. During winter when the lake is covered by ice, this species reproduces sexually, reflecting a high metabolic activity. Throughout the year, L. baicalensis lives in association with dinoflagellates, which - according to the data presented herein - are symbiotic. The dinoflagellates have been determined on the basis of their rDNA/ITS characteristics and were found to display high sequence similarity to Gymnodinium sanguineum. The dinoflagellates give the sponge its characteristic green color, reflecting the high chlorophyll content (chlorophyll-a content in March and September of 3.2 +/- 0.6 microg/g and 1.9 +/- 0.5 microg/g of protein, respectively). With the in vitro cell culture system for sponges, the primmorphs, it could be demonstrated that [(14)C] glycerol is readily taken up by sponge cells; this process can be inhibited by phloretin, an aquaporin channel blocker. In order to prove the effect of cholesterol on the intermediate metabolism of the sponge cells, molecule probes, cDNAs for key enzymes in gluconeogenesis, glycolysis, and citric acid, have been applied in Northern blot studies. The data revealed that the genes coding for the enzymes citrate synthase and fructose-1,6-bisphosphatase are strongly upregulated after exposure of primmorphs to glycerol. This effect is abolished by phloretin. The genes encoding the phosphoglucose isomerase and pyruvate dehydrogenase do not respond to glycerol supply, suggesting that their expression is not under genetic control in L. baicalensis. To prove the assumption that the aquaporin channel is involved in the influx of glycerol in sponge cells, this cDNA was cloned and applied for in situ hybridization studies. The results obtained show that cells surrounding the dinoflagellates become brightly stained after hybridization with the aquaporin this probe. This demonstrates that L. baicalensis cells respond

  6. Aquaporins in Health and Disease: An Overview Focusing on the Gut of Different Species

    Science.gov (United States)

    Pelagalli, Alessandra; Squillacioti, Caterina; Mirabella, Nicola; Meli, Rosaria

    2016-01-01

    Aquaporins (AQPs) play a pivotal role in gut homeostasis since their distribution and function is modulated both in physiological and in pathophysiological conditions. The transport of water and solutes through gut epithelia is essential for osmoregulation and digestive and absorptive functions. This passage is regulated by different AQP isoforms and characterized by their peculiar distribution in the gastrointestinal tract. To date, AQP localization has been identified in the gut and associated organs of several mammalian species by different techniques (immunohistochemical, western blotting, and RT-PCR). The present review describes the modulation of AQP expression, distribution, and function in gut pathophysiology. At the same time, the comparative description of AQP in animal species sheds light on the full range of AQP functions and the screening of their activity as transport modulators, diagnostic biomarkers, and drug targets. Moreover, the phenotype of knockout mice for several AQPs and their compensatory role and the use of specific AQP inhibitors have been also reviewed. The reported data could be useful to design future research in both basic and clinical fields. PMID:27472320

  7. Aquaporins in Health and Disease: An Overview Focusing on the Gut of Different Species

    Directory of Open Access Journals (Sweden)

    Alessandra Pelagalli

    2016-07-01

    Full Text Available Aquaporins (AQPs play a pivotal role in gut homeostasis since their distribution and function is modulated both in physiological and in pathophysiological conditions. The transport of water and solutes through gut epithelia is essential for osmoregulation and digestive and absorptive functions. This passage is regulated by different AQP isoforms and characterized by their peculiar distribution in the gastrointestinal tract. To date, AQP localization has been identified in the gut and associated organs of several mammalian species by different techniques (immunohistochemical, western blotting, and RT-PCR. The present review describes the modulation of AQP expression, distribution, and function in gut pathophysiology. At the same time, the comparative description of AQP in animal species sheds light on the full range of AQP functions and the screening of their activity as transport modulators, diagnostic biomarkers, and drug targets. Moreover, the phenotype of knockout mice for several AQPs and their compensatory role and the use of specific AQP inhibitors have been also reviewed. The reported data could be useful to design future research in both basic and clinical fields.

  8. Pregnant phenotype in aquaporin 8-deficient mice

    Institute of Scientific and Technical Information of China (English)

    Xiao-yan SHA; Zheng-fang XIONG; Hui-shu LIU; Zheng ZHENG; Tong-hui MA

    2011-01-01

    Aim: Aquaporin 8 (AQP8) is expressed within the female reproductive system but its physiological function reminds to be elucidated.This study investigates the role of AQP8 during pregnancy using AQP8-knockout (AQP8-KO) mice.Methods: Homozygous AQP8-KO mice were mated, and the conception rate was recorded. AQP8-KO pregnant mice or their offspring were divided into 5 subgroups according to fetal gestational day (7, 13, 16, 18 GD) and newborn. Wild type C57 pregnant mice served as the control group. The number of pregnant mice, total embryos and atrophic embryos, as well as fetal weight, placental weight and placental area were recorded for each subgroup. The amount of amniotic fluid in each sac at 13, 16, and 18 GD was calculated. Statistical significance was determined by analysis of variance of factorial design and chi-square tests.Results: Conception rates did not differ significantly between AQP8-KO and wild type mice. AQP8-KO pregnant mice had a significantly higher number of embryos compared to wild type controls. Fetal/neonatal weight was also significantly greater in the AQP8-KO group compared to age-matched wild type controls. The amount of amniotic fluid was greater in AQP8-KO pregnant mice than wild type controis, although the FM/AFA (fetal weight/amniotic fluid amount) did not differ. While AQP8-KO placental weight was significantly larger than wild type controls, there was no evidence of placental pathology in either group.Conclusion: The results suggest that AQP8 deficiency plays an important role in pregnancy outcome.

  9. Altered aquaporin expression in glaucoma eyes.

    Science.gov (United States)

    Tran, Thuy Linh; Bek, Toke; la Cour, Morten; Nielsen, Søren; Prause, Jan Ulrik; Hamann, Steffen; Heegaard, Steffen

    2014-09-01

    Aquaporins (AQP) are channels in the cell membrane that mainly facilitate a passive transport of water. In the eye, AQPs are expressed in the ciliary body and retina and may contribute to the pathogenesis of glaucoma and optic neuropathy. We investigated the expression of AQP1, AQP3, AQP4, AQP5, AQP7 and AQP9 in human glaucoma eyes compared with normal eyes. Nine glaucoma eyes were examined. Of these, three eyes were diagnosed with primary open angle glaucoma; three eyes had neovascular glaucoma; and three eyes had chronic angle-closure glaucoma. Six eyes with normal intraocular pressure and without glaucoma were used as control. Immunohistochemistry was performed using antibodies against AQP1, AQP3, AQP4, AQP5, AQP7 and AQP9. For each specimen, optical densities of immunoprecipitates were measured using Photoshop and the staining intensities were calculated. Immunostaining showed labelling of AQP7 and AQP9 in the nonpigmented ciliary epithelium and the staining intensities were significantly decreased in glaucoma eyes (p = 0.003; p = 0.018). AQP7 expression in the Müller cell endfeet was increased (p = 0.046), and AQP9 labelling of the retinal ganglion cells (RGC) showed decreased intensity (p = 0.037). No difference in AQP1, AQP4 and AQP9 expression was found in the optic nerve fibres. This study is the first investigating AQPs in human glaucoma eyes. We found a reduced expression of AQP9 in the retinal ganglion cells of glaucoma eyes. Glaucoma also induced increased AQP7 expression in the Müller cell endfeet. In the ciliary body of glaucoma eyes, the expression of AQP7 and AQP9 was reduced. Therefore, the expression of AQPs seems to play a role in glaucoma.

  10. Aquaporin-4 expression in post-traumatic syringomyelia.

    Science.gov (United States)

    Hemley, Sarah J; Bilston, Lynne E; Cheng, Shaokoon; Chan, Jing Ning; Stoodley, Marcus A

    2013-08-15

    Aquaporin-4 (AQP4) is an astroglial water channel protein that plays an important role in the transmembrane movement of water within the central nervous system. AQP4 has been implicated in numerous pathological conditions involving abnormal fluid accumulation, including spinal cord edema following traumatic injury. AQP4 has not been studied in post-traumatic syringomyelia, a condition that cannot be completely explained by current theories of cerebrospinal fluid dynamics. Alterations of AQP4 expression or function may contribute to the fluid imbalance leading to syrinx formation or enlargement. The aim of this study was to examine AQP4 expression levels and distribution in an animal model of post-traumatic syringomyelia. Immunofluorescence and western blotting were used to assess AQP4 and glial fibrillary acidic protein (GFAP) expression in an excitotoxic amino acid/arachnoiditis model of post-traumatic syringomyelia in Sprague-Dawley rats. At all time-points, GFAP-positive astrocytes were observed in tissue surrounding syrinx cavities, although western blot analysis demonstrated an overall decrease in GFAP expression, except at the latest stage investigated. AQP4 expression was significantly higher at the level of syrinx at three and six weeks following the initial syrinx induction surgery. Significant increases in AQP4 expression also were observed in the upper cervical cord, rostral to the syrinx except in the acute stage of the condition at the three-day time-point. Immunostaining showed that AQP4 was expressed around all syrinx cavities, most notably adjacent to a mature syrinx (six- and 12-week time-point). This suggests a relationship between AQP4 and fluid accumulation in post-traumatic syringomyelia. However, whether this is a causal relationship or occurs in response to an increase in fluid needs to be established.

  11. p53 isoform profiling in glioblastoma and injured brain.

    Science.gov (United States)

    Takahashi, R; Giannini, C; Sarkaria, J N; Schroeder, M; Rogers, J; Mastroeni, D; Scrable, H

    2013-06-27

    The tumor suppressor p53 has been found to be the most commonly mutated gene in human cancers; however, the frequency of p53 mutations varies from 10 to 70% across different cancer types. This variability can partly be explained by inactivating mechanisms aside from direct genomic polymorphisms. The p53 gene encodes 12 isoforms, some of which can modulate full-length p53 activity in cancer. In this study, we characterized p53 isoform expression patterns in glioblastoma, gliosis, non-tumor brain and neural progenitor cells by SDS-PAGE, immunoblot, mass spectrometry and reverse transcription-PCR. We found that the most consistently expressed isoform in glioblastoma, Δ40p53, was uniquely expressed in regenerative processes, such as those involving neural progenitor cells and gliosis compared with tumor samples. Isoform profiling of glioblastoma tissues revealed the presence of both Δ40p53 and full-length p53, neither of which were detected in non-tumor cerebral cortex. Upon xenograft propagation of tumors, p53 levels increased. The variability of overall p53 expression and relative levels of isoforms suggest fluctuations in subpopulations of cells with greater or lesser capacity for proliferation, which can change as the tumor evolves under different growth conditions.

  12. Aquaporins in sperm osmoadaptation: an emerging role for volume regulation

    Institute of Scientific and Technical Information of China (English)

    Qi CHEN; En-kui DUAN

    2011-01-01

    Upon ejaculation, mammalian sperm experience a natural osmotic decrease during male to female reproductive tract transition. This hypo-osmotic exposure not only activates sperm motility, but also poses potential harm to sperm structure and function by inducing unwanted cell swelling. In this physiological context, regulatory volume decrease (RVD) is the major mechanism that protects cells from detrimental swelling, and is essential to sperm survival and normal function. Aquaporins are selective water channels that enable rapid water transport across cell membranes. Aquaporins have been implicated in sperm osmoregulation. Recent discoveries show that Aquaporin-3 (AQP3), a water channel protein, is localized in sperm tail membranes and that AQP3 mutant sperm show defects in volume regulation and excessive cell swelling upon physiological hypotonic stress in the female reproductive tract, thereby highlighting the importance of AQP3 in the postcopulatory sperm RVD process. In this paper, we discuss current knowledge, remaining questions and hypotheses about the function and mechanismic basis of aquaporins for volume regulation in sperm and other cell types.

  13. Water transport between CNS compartments: contributions of aquaporins and cotransporters

    DEFF Research Database (Denmark)

    MacAulay, N; Zeuthen, T

    2010-01-01

    or hydrocephalus. The molecular pathways by which water molecules cross the cell membranes of the brain are not well-understood, although the discovery of aquaporin 4 (AQP4) in the brain improved our understanding of some of these transport processes, particularly under pathological conditions. In the present...

  14. Crystal Structure of an Ammonia-Permeable Aquaporin.

    Directory of Open Access Journals (Sweden)

    Andreas Kirscht

    2016-03-01

    Full Text Available Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.

  15. Bladder function impairment in aquaporin-2 defective nephrogenic diabetes insipidus.

    NARCIS (Netherlands)

    Shalev, H.; Romanovsky, I.; Knoers, N.V.A.M.; Lupa, S.; Landau, D.

    2004-01-01

    BACKGROUND: The aim of this study was to describe the urological complications associated with nephrogenic diabetes insipidus (NDI) due to a mutation in aquaporin-2 (AQP2), a collecting-duct protein activated by ADH signalling. METHODS: We provide a case series description of a group of seven patien

  16. Crystal Structure of an Ammonia-Permeable Aquaporin.

    Science.gov (United States)

    Kirscht, Andreas; Kaptan, Shreyas S; Bienert, Gerd Patrick; Chaumont, François; Nissen, Poul; de Groot, Bert L; Kjellbom, Per; Gourdon, Pontus; Johanson, Urban

    2016-03-01

    Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.

  17. Aquaporin Inhibition by Gold(III) Compounds : New Insights

    NARCIS (Netherlands)

    Martins, Ana Paula; Ciancetta, Antonella; Batista de Almeida, Andreia; Marrone, Alessandro; Re, Nazzareno; Soveral, Graca; Casini, Angela

    2013-01-01

    Aquaporins (AQPs) are membrane water/glycerol channels with essential roles in biological systems, as well as being promising targets for therapy and imaging. Using a stopped-flow method, a series of gold(III), platinum(II) and copper(II) complexes bearing nitrogen donor ligands, such as 1,10-phenat

  18. Human Aquaporin-4 and Molecular Modeling: Historical Perspective and View to the Future

    Science.gov (United States)

    Mangiatordi, Giuseppe Felice; Alberga, Domenico; Trisciuzzi, Daniela; Lattanzi, Gianluca; Nicolotti, Orazio

    2016-01-01

    Among the different aquaporins (AQPs), human aquaporin-4 (hAQP4) has attracted the greatest interest in recent years as a new promising therapeutic target. Such a membrane protein is, in fact, involved in a multiple sclerosis-like immunopathology called Neuromyelitis Optica (NMO) and in several disorders resulting from imbalanced water homeostasis such as deafness and cerebral edema. The gap of knowledge in its functioning and dynamics at the atomistic level of detail has hindered the development of rational strategies for designing hAQP4 modulators. The application, lately, of molecular modeling has proved able to fill this gap providing a breeding ground to rationally address compounds targeting hAQP4. In this review, we give an overview of the important advances obtained in this field through the application of Molecular Dynamics (MD) and other complementary modeling techniques. The case studies presented herein are discussed with the aim of providing important clues for computational chemists and biophysicists interested in this field and looking for new challenges. PMID:27420052

  19. Human Aquaporin-4 and Molecular Modeling: Historical Perspective and View to the Future

    Directory of Open Access Journals (Sweden)

    Giuseppe Felice Mangiatordi

    2016-07-01

    Full Text Available Among the different aquaporins (AQPs, human aquaporin-4 (hAQP4 has attracted the greatest interest in recent years as a new promising therapeutic target. Such a membrane protein is, in fact, involved in a multiple sclerosis-like immunopathology called Neuromyelitis Optica (NMO and in several disorders resulting from imbalanced water homeostasis such as deafness and cerebral edema. The gap of knowledge in its functioning and dynamics at the atomistic level of detail has hindered the development of rational strategies for designing hAQP4 modulators. The application, lately, of molecular modeling has proved able to fill this gap providing a breeding ground to rationally address compounds targeting hAQP4. In this review, we give an overview of the important advances obtained in this field through the application of Molecular Dynamics (MD and other complementary modeling techniques. The case studies presented herein are discussed with the aim of providing important clues for computational chemists and biophysicists interested in this field and looking for new challenges.

  20. Cloning and sequence analysis of gene encoding plasma aquaporin of Tamarix albiflonum

    Institute of Scientific and Technical Information of China (English)

    DONG Yuzhi; YANG Chuanping; ZHANG Daoyuan; WANG Yucheng

    2007-01-01

    Plant aquaporins are water-selected-channels in plants and are involved in seed germination,cell elongation,stoma movement,fertilization and so on.Some plant aquapotins also play an important role in drought stress response.In this paper,the gene encoding the Tamarix albiflonum Aquaporin (AQP) was amplified by 5'rapid amplification of cDNA end (RACE) on the basis of the sequence information obtained from the expressed sequence tag of the subtractive hybridization library constructed under PEG6000 stress.The cDNA of the T.albiflonum AQP gene is 1,043 bp long,encoding a protein of 287 amino acids with a predicted molecular mass of 30.9 kDa,has 6 transmembrane regions,and possessing the major intrinsic protein (MIP) family signal consensus sequence SGXHXNPAVT and the higher plant plasma membrane intrinsic protein (PIP) highly conservative sequence GGGANXXXXGY and TGI/TNPARSL /FGAA I/VI/VF/YN.A comparative molecular analysis of the nucleotide sequence in National Center for Biotechnology Information (NCBI) databases showed that it shared 95% homology with the gene ofArabidopsis thaliana (MIP-C),with a theoretical isoelectric point 8.84.

  1. Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

    Directory of Open Access Journals (Sweden)

    Barbara Arbeithuber

    2015-01-01

    Full Text Available Several studies have revealed that aquaporins play a role in tumor progression and invasion. In breast carcinomas, high levels of aquaporin 5 (AQP5, a membrane protein involved in water transport, have been linked to increased cell proliferation and migration, thus facilitating tumor progression. Despite the potential role of AQP5 in mammary oncogenesis, the mechanisms controlling mammary AQP5 expression are poorly understood. In other tissues, AQP5 expression has been correlated with its promoter methylation, yet, very little is known about AQP5 promoter methylation in the mammary gland. In this work, we used the mouse mammary gland cell line EpH4, in which we controlled AQP5 expression via the steroid hormone dexamethasone (Dex to further investigate mechanisms regulating AQP5 expression. In this system, we observed a rapid drop of AQP5 mRNA levels with a delay of several hours in AQP5 protein, suggesting transcriptional control of AQP5 levels. Yet, AQP5 expression was independent of its promoter methylation, or to the presence of negative glucocorticoid receptor elements (nGREs in its imminent promoter region, but was rather influenced by the cell proliferative state or cell density. We conclude that AQP5 promoter methylation is not a universal mechanism for AQP5 regulation and varies on cell and tissue type.

  2. Recombinant production of human Aquaporin-1 to an exceptional high membrane density in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Julie Bomholt

    Full Text Available In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tagged with yeast enhanced GFP for quantification of functional expression, determination of sub-cellular localization, estimation of in vivo folding efficiency and establishment of a purification protocol. Aquaporin-1 was found to constitute 8.5 percent of total membrane protein content after expression at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction of the expression temperature to 15°C almost completely prevented Aquaporin-1 mal-folding. Bioimaging of live yeast cells revealed that recombinant Aquaporin-1 accumulated in the yeast plasma membrane. A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1 and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes.

  3. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Helix Nielsen, Claus; Scharff-Poulsen, Peter

    2013-01-01

    In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tag......In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C...... at 15°C in a yeast host over-producing the Gal4p transcriptional activator and growth in amino acid supplemented minimal medium. In-gel fluorescence combined with western blotting showed that low accumulation of correctly folded recombinant Aquaporin-1 at 30°C was due to in vivo mal-folding. Reduction...... of the expression temperature to 15°C almost completely prevented Aquaporin-1 mal-folding. Bioimaging of live yeast cells revealed that recombinant Aquaporin-1 accumulated in the yeast plasma membrane. A detergent screen for solubilization revealed that CYMAL-5 was superior in solubilizing recombinant Aquaporin-1...

  4. Normal Immunostaining Pattern for Aquaporin-5 in the Lesions of Palmoplantar Hyperhidrosis

    Directory of Open Access Journals (Sweden)

    Kyoko Nakahigashi

    2013-03-01

    Full Text Available Aquaporin-5 (AQP-5 is a water-transporting protein expressed in mammal sweat glands. It has been reported that the expression of AQP-5 is involved in sweating of mice, rats, and horses. However, the physiological function of human AQP-5 is still uncertain. In this report, we examined the expression pattern of AQP-5 in the skin lesions of palmoplantar hyperhidrosis in patients with Nagashima-type palmoplantar hyperkeratosis (PPK. We found that there was no significant difference in AQP-5 expression in the palmoplantar skin of healthy subjects and patients with palmoplantar hyperhidrosis. Our findings suggest that a mechanism other than AQP-5 may be involved in the pathogenesis of hyperhidrosis in PPK.

  5. Characterization of Leishmania donovani aquaporins shows presence of subcellular aquaporins similar to tonoplast intrinsic proteins of plants.

    Directory of Open Access Journals (Sweden)

    Neha Biyani

    Full Text Available Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs annotated as AQP9 (230aa, AQP putative (294aa, AQP-like protein (279aa, AQP1 (314aa and AQP-like protein (596aa. We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis

  6. Inference of Isoforms from Short Sequence Reads

    Science.gov (United States)

    Feng, Jianxing; Li, Wei; Jiang, Tao

    Due to alternative splicing events in eukaryotic species, the identification of mRNA isoforms (or splicing variants) is a difficult problem. Traditional experimental methods for this purpose are time consuming and cost ineffective. The emerging RNA-Seq technology provides a possible effective method to address this problem. Although the advantages of RNA-Seq over traditional methods in transcriptome analysis have been confirmed by many studies, the inference of isoforms from millions of short sequence reads (e.g., Illumina/Solexa reads) has remained computationally challenging. In this work, we propose a method to calculate the expression levels of isoforms and infer isoforms from short RNA-Seq reads using exon-intron boundary, transcription start site (TSS) and poly-A site (PAS) information. We first formulate the relationship among exons, isoforms, and single-end reads as a convex quadratic program, and then use an efficient algorithm (called IsoInfer) to search for isoforms. IsoInfer can calculate the expression levels of isoforms accurately if all the isoforms are known and infer novel isoforms from scratch. Our experimental tests on known mouse isoforms with both simulated expression levels and reads demonstrate that IsoInfer is able to calculate the expression levels of isoforms with an accuracy comparable to the state-of-the-art statistical method and a 60 times faster speed. Moreover, our tests on both simulated and real reads show that it achieves a good precision and sensitivity in inferring isoforms when given accurate exon-intron boundary, TSS and PAS information, especially for isoforms whose expression levels are significantly high.

  7. Expression of phosphoinositide-specific phospholipase C isoforms in native endothelial cells.

    Directory of Open Access Journals (Sweden)

    Delphine M Béziau

    Full Text Available Phospholipase C (PLC comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA, pulmonary (PA and middle cerebral arteries (MCA. mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA, δ4 (only expressed in MCA, η1 (expressed in all but MA and ζ (not detected in any vascular beds tested. The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1 in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found

  8. Effects of Proteoliposome Composition and Draw Solution Types on Separation Performance of Aquaporin-Based Proteoliposomes

    DEFF Research Database (Denmark)

    Zhao, Yang; Vararattanavech, Ardcharaporn; Li, Xuesong;

    2013-01-01

    Aquaporins are a large family of water transport proteins in cell membranes. Their high water permeability and solute rejection make them potential building blocks for high-performance biomimetic membranes for desalination. In the current study, proteoliposomes were prepared using AquaporinZ from...... dissolved ions in seawater (e.g., Mg2+ and SO42–) on the stability of proteoliposomes, and design criteria for aquaporin-based biomimetic membranes are proposed in the context of desalination....

  9. Root aquaporins contribute to whole plant water fluxes under drought stress in rice (Oryza sativa L.).

    Science.gov (United States)

    Grondin, Alexandre; Mauleon, Ramil; Vadez, Vincent; Henry, Amelia

    2016-02-01

    Aquaporin activity and root anatomy may affect root hydraulic properties under drought stress. To better understand the function of aquaporins in rice root water fluxes under drought, we studied the root hydraulic conductivity (Lpr) and root sap exudation rate (Sr) in the presence or absence of an aquaporin inhibitor (azide) under well-watered conditions and following drought stress in six diverse rice varieties. Varieties varied in Lpr and Sr under both conditions. The contribution of aquaporins to Lpr was generally high (up to 79% under well-watered conditions and 85% under drought stress) and differentially regulated under drought. Aquaporin contribution to Sr increased in most varieties after drought, suggesting a crucial role for aquaporins in osmotic water fluxes during drought and recovery. Furthermore, root plasma membrane aquaporin (PIP) expression and root anatomical properties were correlated with hydraulic traits. Three chromosome regions highly correlated with hydraulic traits of the OryzaSNP panel were identified, but did not co-locate with known aquaporins. These results therefore highlight the importance of aquaporins in the rice root radial water pathway, but emphasize the complex range of additional mechanisms related to root water fluxes and drought response.

  10. Genome-wide identification and expression analysis of aquaporins in tomato.

    Science.gov (United States)

    Reuscher, Stefan; Akiyama, Masahito; Mori, Chiharu; Aoki, Koh; Shibata, Daisuke; Shiratake, Katsuhiro

    2013-01-01

    The family of aquaporins, also called water channels or major intrinsic proteins, is characterized by six transmembrane domains that together facilitate the transport of water and a variety of low molecular weight solutes. They are found in all domains of life, but show their highest diversity in plants. Numerous studies identified aquaporins as important targets for improving plant performance under drought stress. The phylogeny of aquaporins is well established based on model species like Arabidopsis thaliana, which can be used as a template to investigate aquaporins in other species. In this study we comprehensively identified aquaporin encoding genes in tomato (Solanum lycopersicum), which is an important vegetable crop and also serves as a model for fleshy fruit development. We found 47 aquaporin genes in the tomato genome and analyzed their structural features. Based on a phylogenetic analysis of the deduced amino acid sequences the aquaporin genes were assigned to five subfamilies (PIPs, TIPs, NIPs, SIPs and XIPs) and their substrate specificity was assessed on the basis of key amino acid residues. As ESTs were available for 32 genes, expression of these genes was analyzed in 13 different tissues and developmental stages of tomato. We detected tissue-specific and development-specific expression of tomato aquaporin genes, which is a first step towards revealing the contribution of aquaporins to water and solute transport in leaves and during fruit development.

  11. Genome-wide identification and expression analysis of aquaporins in tomato.

    Directory of Open Access Journals (Sweden)

    Stefan Reuscher

    Full Text Available The family of aquaporins, also called water channels or major intrinsic proteins, is characterized by six transmembrane domains that together facilitate the transport of water and a variety of low molecular weight solutes. They are found in all domains of life, but show their highest diversity in plants. Numerous studies identified aquaporins as important targets for improving plant performance under drought stress. The phylogeny of aquaporins is well established based on model species like Arabidopsis thaliana, which can be used as a template to investigate aquaporins in other species. In this study we comprehensively identified aquaporin encoding genes in tomato (Solanum lycopersicum, which is an important vegetable crop and also serves as a model for fleshy fruit development. We found 47 aquaporin genes in the tomato genome and analyzed their structural features. Based on a phylogenetic analysis of the deduced amino acid sequences the aquaporin genes were assigned to five subfamilies (PIPs, TIPs, NIPs, SIPs and XIPs and their substrate specificity was assessed on the basis of key amino acid residues. As ESTs were available for 32 genes, expression of these genes was analyzed in 13 different tissues and developmental stages of tomato. We detected tissue-specific and development-specific expression of tomato aquaporin genes, which is a first step towards revealing the contribution of aquaporins to water and solute transport in leaves and during fruit development.

  12. Identification and characterization of plasma membrane aquaporins isolated from fiber cells of Calotropis procera

    Institute of Scientific and Technical Information of China (English)

    Usman ASLAM; Asia KHATOON; Hafiza Masooma Naseer CHEEMA; Aftab BASHIR

    2013-01-01

    Calotropis procera,commonly known as "milkweed",possesses long seed trichomes for seed dispersal and has the ability to survive under harsh conditions such as drought and salinity.Aquaporins are water channel proteins expressed in all land plants,divided into five subfamilies plasma membrane intrinsic proteins (PIPs),tonoplast intrinsic proteins (TIPs),NOD26-1ike proteins (NIPs),small basic intrinsic proteins (SIPs),and the unfamiliar X intrinsic proteins (XlPs).PIPs constitute the largest group of water channel proteins that are involved in different developmental and regulatory mechanisms including water permeability,cell elongation,and stomata opening.Aquaporins are also involved in abiotic stress tolerance and cell expansion mechanisms,but their role in seed trichomes (fiber cells) has never been investigated.A large number of clones isolated from C.procera fiber cDNA library showed sequence homology to PIPs.Both expressed sequence tags (ESTs) and real-time polymerase chain reaction (PCR) studies revealed that the transcript abundance of this gene family in fiber cells of C.procera is greater than that of cotton.Full-length cDNAs of CpPIP1 and CpPIP2 were isolated from C.procera fiber cDNA library and used for constructing plant expression vectors under constitutive (2x35S) and trichome-specific (GhLTP3) promoters.Transgenic tobacco plants were developed via Agrobacterium-mediated transformation.The phenotypic characteristics of the plants were observed after confirming the integration of transgene in plants.It was observed that CpPIP2 expression cassette under 2x35S and GhLTP3 promoter enhanced the numbers of stem and leave trichomes.However,2x35S::CpPIP2 has a more amplified effect on trichome density and length than GhLTP3::CpPIP2 and other PIP constructs.These findings imply the role of C.procera PIP aquaporins in fiber cell elongation.The PIPs-derived cell expansion mechanism may be exploited through transgenic approaches for improvement of fiber staple

  13. Electrostatic tuning of permeation and selectivity in aquaporin water channels

    DEFF Research Database (Denmark)

    Jensen, Mogens O Stibius; Tajkhorshid, E.; Schulten, K.

    2003-01-01

    -palmitoyloleylphosphatidylethanolamine membrane was used for the simulations. During the simulations, water molecules pass through the channel in single file. The movement of the single. le water molecules through the channel is concerted, and we show that it can be described by a continuous-time random-walk model. The integrity......Water permeation and electrostatic interactions between water and channel are investigated in the Escherichia coli glycerol uptake facilitator GlpF, a member of the aquaporin water channel family, by molecular dynamics simulations. A tetrameric model of the channel embedded in a 16:0/ 18:1c9...... of the single file remains intact during the permeation, indicating that a disrupted water chain is unlikely to be the mechanism of proton exclusion in aquaporins. Specific hydrogen bonds between permeating water and protein at the channel center (at two conserved Asp-Pro-Ala "NPA'' motifs), together...

  14. Aquaporin-Based Biomimetic Polymeric Membranes: Approaches and Challenges

    Directory of Open Access Journals (Sweden)

    Joachim Habel

    2015-07-01

    Full Text Available In recent years, aquaporin biomimetic membranes (ABMs for water separation have gained considerable interest. Although the first ABMs are commercially available, there are still many challenges associated with further ABM development. Here, we discuss the interplay of the main components of ABMs: aquaporin proteins (AQPs, block copolymers for AQP reconstitution, and polymer-based supporting structures. First, we briefly cover challenges and review recent developments in understanding the interplay between AQP and block copolymers. Second, we review some experimental characterization methods for investigating AQP incorporation including freeze-fracture transmission electron microscopy, fluorescence correlation spectroscopy, stopped-flow light scattering, and small-angle X-ray scattering. Third, we focus on recent efforts in embedding reconstituted AQPs in membrane designs that are based on conventional thin film interfacial polymerization techniques. Finally, we describe some new developments in interfacial polymerization using polyhedral oligomeric silsesquioxane cages for increasing the physical and chemical durability of thin film composite membranes.

  15. Bacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus.

    Directory of Open Access Journals (Sweden)

    Apiruck Watthanasurorot

    2011-06-01

    Full Text Available The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam, encodes 9(Ig-4(FNIII-(Ig-2(FNIII-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish.

  16. Overexpression of Aquaporin-1 in lung adenocarcinomas and pleural mesotheliomas

    OpenAIRE

    López-Campos, José Luis; Sánchez Silva, Rocío; Gómez Izquierdo, Lourdes; Márquez, Eduardo; Ortega Ruiz, Francisco; Cejudo, Pilar; Barrot Cortés, Emilia; Toledo Aral, Juan José; Echevarría, Miriam

    2011-01-01

    Aquaporin-1 (AQP1) is the main water channel responsible for water transport through many epithelia and endothelia. The latest evidence pointed toward an important role of this protein also in gas permeation, angiogenesis, cell proliferation and migration. In the present work we studied the expression of AQP1 by immunohistochemical staining of 92 lung biopsies from patients diagnosed with a pleuropulmonary tumor (71 lung and 21 pleural neoplasms). AQP1 expression wa...

  17. Aquaporin-Based Biomimetic Polymeric Membranes: Approaches and Challenges

    DEFF Research Database (Denmark)

    Habel, Joachim Erich Otto; Hansen, Michael; Kynde, Søren;

    2015-01-01

    In recent years, aquaporin biomimetic membranes (ABMs) for water separation have gained considerable interest. Although the first ABMs are commercially available, there are still many challenges associated with further ABM development. Here, we discuss the interplay of the main components of ABMs...... thin film interfacial polymerization techniques. Finally, we describe some new developments in interfacial polymerization using polyhedral oligomeric silsesquioxane cages for increasing the physical and chemical durability of thin film composite membranes....

  18. Vasopressin-dependent short-term regulation of aquaporin 4 expressed in Xenopus oocytes

    DEFF Research Database (Denmark)

    Moeller, H B; Fenton, R A; Zeuthen, T;

    2009-01-01

    following pathologies such as brain injuries, brain tumours, and cerebral ischemia. As vasopressin and its G-protein-coupled receptor (V1(a)R) have been shown to affect the outcome of brain edema, we have investigated the regulatory interaction between AQP4 and V1(a)R by heterologous expression in Xenopus......Aquaporin 4 (AQP4) is abundantly expressed in the perivascular glial endfeet in the central nervous system (CNS), where it is involved in the exchange of fluids between blood and brain. At this location, AQP4 contributes to the formation and/or the absorption of the brain edema that may arise......)R may prove to be a potential therapeutic target in the prevention and treatment of brain edema....

  19. Trypanosomatid Aquaporins: Roles in Physiology and Drug Response

    Directory of Open Access Journals (Sweden)

    Goutam Mandal

    2013-12-01

    Full Text Available In the class Kinetoplastida, we find an order of parasitic protozoans classified as Trypanosomatids. Three major pathogens form part of this order, Trypanosoma cruzi, Trypanosoma brucei, and Leishmania, which are responsible for disease and fatalities in millions of humans worldwide, especially in non-industrialized countries in tropical and sub-tropical regions. In order to develop new drugs and treatments, the physiology of these pathogenic protozoans has been studied in detail, specifically the significance of membrane transporters in host parasites interactions. Aquaporins and Aquaglyceroporins (AQPs are a part of the major intrinsic proteins (MIPs super-family. AQPs are characterized for their ability to facilitate the diffusion of water (aquaporin, glycerol (aquaglyceroporin, and other small-uncharged solutes. Furthermore, AQPs have been shown to allow the ubiquitous passage of some metalloids, such as trivalent arsenic and antimony. These trivalent metalloids are the active ingredient of a number of chemotherapeutic agents used against certain cancers and protozoan parasitic infections. Recently, the importance of the AQPs not only in osmotic adaptations but also as a factor in drug resistance of the trypanosomatid parasites has been reported. In this review, we will describe the physiological functions of aquaporins and their effect in drug response across the different trypanosomatids.

  20. An aquaporin PvTIP4;1 from Pteris vittata may mediate arsenite uptake.

    Science.gov (United States)

    He, Zhenyan; Yan, Huili; Chen, Yanshan; Shen, Hongling; Xu, Wenxiu; Zhang, Haiyan; Shi, Lei; Zhu, Yong-Guan; Ma, Mi

    2016-01-01

    The fern Pteris vittata is an arsenic hyperaccumulator. The genes involved in arsenite (As(III)) transport are not yet clear. Here, we describe the isolation and characterization of a new P. vittata aquaporin gene, PvTIP4;1, which may mediate As(III) uptake. PvTIP4;1 was identified from yeast functional complement cDNA library of P. vittata. Arsenic toxicity and accumulating activities of PvTIP4;1 were analyzed in Saccharomyces cerevisiae and Arabidopsis. Subcellular localization of PvTIP4;1-GFP fusion protein in P. vittata protoplast and callus was conducted. The tissue expression of PvTIP4;1 was investigated by quantitative real-time PCR. Site-directed mutagenesis of the PvTIP4;1 aromatic/arginine (Ar/R) domain was studied. Heterologous expression in yeast demonstrates that PvTIP4;1 was able to facilitate As(III) diffusion. Transgenic Arabidopsis showed that PvTIP4;1 increases arsenic accumulation and induces arsenic sensitivity. Images and FM4-64 staining suggest that PvTIP4;1 localizes to the plasma membrane in P. vittata cells. A tissue location study shows that PvTIP4;1 transcripts are mainly expressed in roots. Site-directed mutation in yeast further proved that the cysteine at the LE1 position of PvTIP4;1 Ar/R domain is a functional site. PvTIP4;1 is a new represented tonoplast intrinsic protein (TIP) aquaporin from P. vittata and the function and location results imply that PvTIP4;1 may be involved in As(III) uptake.

  1. Two isoforms of trimming glucosidase II exist in mammalian tissues and cell lines but not in yeast and insect cells.

    Science.gov (United States)

    Ziak, M; Meier, M; Etter, K S; Roth, J

    2001-01-12

    We previously cloned glucosidase II and provided in vivo evidence for its involvement in protein folding quality control. DNA-sequencing of different clones demonstrated the existence of two isoforms of glucosidase II which differed by 66 nucleotides due to alternative splicing. The existence of two enzyme isoforms in various organs of pig and rat as well as human, bovine, rat, and mouse cell lines could be demonstrated by RT-PCR and Western blotting. Furthermore, the two isoforms of glucosidase II could be detected in embryonic and postnatal rat kidney and liver. In yeast, Saccharomyces cerevisiae, and in insects, Drosophila S2 cells, only one isoforms of the enzyme was detectable. The ubiquitous occurrence of the two glucosidase II isoforms in mammalian tissues and cell lines might be indicative of a special function of each isoform.

  2. Mast cells express novel functional IL-15 receptor alpha isoforms.

    Science.gov (United States)

    Bulanova, Elena; Budagian, Vadim; Orinska, Zane; Krause, Hans; Paus, Ralf; Bulfone-Paus, Silvia

    2003-05-15

    Mast cells previously have been reported to be regulated by IL-15 and to express a distinct IL-15R, termed IL-15RX. To further examine IL-15 binding and signaling in mast cells, we have studied the nature of the IL-15R and some of its biological activities in these cells. In this study, we report the existence of three novel isoforms of the IL-15R alpha chain in murine bone marrow-derived mast cells as a result of an alternative exon-splicing mechanism within the IL-15R alpha gene. These correspond to new mRNA transcripts lacking exon 4; exons 3 and 4; or exons 3, 4, and 5 (IL-15R alpha Delta 4, IL-15R alpha Delta 3,4, IL-15R alpha Delta 3,4,5). After transient transfection in COS-7 cells, all IL-15R alpha isoforms associate with the Golgi apparatus, the endoplasmic reticulum, the perinuclear space, and the cell membrane. Analysis of glycosylation pattern demonstrates the usage of a single N-glycosylation site, while no O-glycosylation is observed. Importantly, IL-15 binds with high affinity to, and promotes the survival of, murine BA/F3 cells stably transfected with the IL-15R alpha isoforms. Furthermore, we report that signaling mediated by IL-15 binding to the newly identified IL-15R alpha isoforms involves the phosphorylation of STAT3, STAT5, STAT6, Janus kinase 2, and Syk kinase. Taken together, our data indicate that murine mast cells express novel, fully functional IL-15R alpha isoforms, which can explain the selective regulatory effects of IL-15 on these cells.

  3. Isolation and characterization of patatin isoforms

    NARCIS (Netherlands)

    Pots, A.M.; Gruppen, H.; Hessing, M.; Boekel, M.A.J.S. van; Voragen, A.G.J.

    1999-01-01

    Patatin has, so far, been considered a homogeneous group of proteins. A comparison of the isoforms in terms of structural properties or stability has not been reported. A method to obtain various isoform fractions as well as a comparison of the physicochemical properties of these pools is presented.

  4. Differences in distribution and regulation of astrocytic aquaporin-4 in human and rat hydrocephalic brain

    DEFF Research Database (Denmark)

    Skjolding, Anders Daehli; Holst, Anders Vedel; Broholm, Helle

    2013-01-01

    findings to human pathophysiology. This study compares expression of aquaporin-4 in hydrocephalic human brain with human controls and hydrocephalic rat brain. Methods:  Cortical biopsies from patients with chronic hydrocephalus (n=29) were sampled secondary to planned surgical intervention. Aquaporin-4...

  5. Regulation of Aquaporin Z osmotic permeability in ABA tri-block copolymer

    Directory of Open Access Journals (Sweden)

    Wenyuan Xie

    2015-08-01

    Full Text Available Aquaporins are transmembrane water channel proteins present in biological plasma membranes that aid in biological water filtration processes by transporting water molecules through at high speeds, while selectively blocking out other kinds of solutes. Aquaporin Z incorporated biomimetic membranes are envisaged to overcome the problem of high pressure needed, and holds great potential for use in water purification processes, giving high flux while keeping energy consumption low. The functionality of aquaporin Z in terms of osmotic permeability might be regulated by factors such as pH, temperature, crosslinking and hydrophobic thickness of the reconstituted bilayers. Hence, we reconstituted aquaporin Z into vesicles that are made from a series of amphiphilic block copolymers PMOXA-PDMS-PMOXAs with various hydrophobic molecular weights. The osmotic permeability of aquaporin Z in these vesicles was determined through a stopped-flow spectroscopy. In addition, the temperature and pH value of the vesicle solutions were adjusted within wide ranges to investigate the regulation of osmotic permeability of aquaporin Z through external conditions. Our results show that aquaporin Z permeability was enhanced by hydrophobic mismatch. In addition, the water filtration mechanism of aquaporin Z is significantly affected by the concentration of H+ and OH- ions.

  6. Aquaporin-2: COOH terminus is necessary but not sufficient for routing to the apical membrane.

    NARCIS (Netherlands)

    Deen, P.M.T.; Balkom, B.W.M. van; Savelkoul, P.J.M.; Kamsteeg, E.J.; Raak, M.M.J.P. van; Jennings, M.L.; Muth, T.R.; Rajendran, V.; Caplan, M.J.

    2002-01-01

    Renal regulation of mammalian water homeostasis is mediated by the aquaporin-1 (AQP1) water channel, which is expressed in the apical and basolateral membranes of proximal tubules and descending limbs of Henle, and aquaporin-2 (AQP2), which is redistributed from intracellular vesicles to the apical

  7. Aquaporin-4 gene silencing protects injured neurons after early cerebral infarction

    Institute of Scientific and Technical Information of China (English)

    Zhan-ping He; Hong Lu

    2015-01-01

    Aquaporin-4 regulates water molecule channels and is important in tissue regulation and water transportation in the brain. Upregulation of aquaporin-4 expression is closely related to cel-lular edema after early cerebral infarction. Cellular edema and aquaporin-4 expression can be determined by measuring cerebral infarct area and apparent diffusion coefficient using diffu-sion-weighted imaging (DWI). We examined the effects of silencing aquaporin-4 on cerebral infarction. Rat models of cerebral infarction were established by occlusion of the right middle cerebral artery and siRNA-aquaporin-4 was immediately injectedvia the right basal ganglia. In control animals, the area of high signal intensity and relative apparent diffusion coefifcient value on T2-weighted imaging (T2WI) and DWI gradually increased within 0.5–6 hours after cerebral infarction. After aquaporin-4 gene silencing, the area of high signal intensity on T2WI and DWI reduced, relative apparent diffusion coefifcient value was increased, and cellular edema was ob-viously alleviated. At 6 hours after cerebral infarction, the apparent diffusion coefifcient value was similar between treatment and model groups, but angioedema was still obvious in the treat-ment group. These results indicate that aquaporin-4 gene silencing can effectively relieve cellular edema after early cerebral infarction; and when conducted accurately and on time, the diffusion coefifcient value and the area of high signal intensity on T2WI and DWI can relfect therapeutic effects of aquaporin-4 gene silencing on cellular edema.

  8. Increased expression of aquaporin-4 in human traumatic brain injury and brain tumors

    Institute of Scientific and Technical Information of China (English)

    HuaHu; Wei-PingZhang; LeiZhang; ZhongChen; Er-QingWei

    2004-01-01

    Aquaporin-4 (AQP4) is one of the aquaporins (AQPs), a water channel family. In the brain, AQP4 is expressed in astroeyte foot processes, and plays an important role in water homeostasis and in the formation of brain edema. In our study, AQP4 expression in human brain specimens from patients with traumatic brain injury or different brain tumors was detected

  9. Dynamic expression and localization of c-MET isoforms in the developing rat pancreas.

    Science.gov (United States)

    Wu, Yulong; Cheng, Mei; Shi, Zhen; Feng, Zhenqing; Guan, Xiaohong

    2014-01-01

    Pancreata from Sprague Dawley rats of different developmental stages were studied to determine the expression and cellular localization of different c-MET isoforms in the developing rat pancreas. Pancreatic mRNA and protein expression levels of c-MET at different developmental stages from embryo to adult were detected by reverse transcription-polymerase chain reaction and by western blotting. To identify the cellular localization of c-MET protein in the developing rat pancreas, double immunofluorescent staining was performed using antibodies for cell type-specific markers and for c-MET. The expression of two isoforms of c-MET (190 kDa and 170 kDa) coincided with the development of the pancreas. The 190 kDa isoform of c-MET is expressed during embryonic stages, and its expression is replaced by the expression of the 170 kDa isoform as the pancreas develops. Only the 170 kDa isoform is expressed in the adult rat pancreas. Throughout all stages of pancreatic development, c-MET is expressed by vimentin-positive cells. In contrast, c-MET staining was stronger in rat pancreata from newborn to adult stages and overlapped with insulin-positive beta-cells. The dynamic expression and localization of different c-MET isoforms in the rat pancreas during different developmental stages indicates that distinct c-MET isoform might be involved in different aspects of pancreatic development.

  10. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    Directory of Open Access Journals (Sweden)

    Angélique Guillaudeau

    Full Text Available The EGFR (epidermal growth factor receptor is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a, normal and tumor cells produce soluble EGFR isoforms (sEGFR that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2, 3 (v3 and 4 (v4 mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab and intracellular domain targeted antibody (ICD-Ab. EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade, histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS. PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types.

  11. Age-related hearing loss: aquaporin 4 gene expression changes in the mouse cochlea and auditory midbrain.

    Science.gov (United States)

    Christensen, Nathan; D'Souza, Mary; Zhu, Xiaoxia; Frisina, Robert D

    2009-02-01

    Presbycusis -- age-related hearing loss, is the number one communication disorder, and one of the top three chronic medical conditions of our aged population. Aquaporins, particularly aquaporin 4 (Aqp4), are membrane proteins with important roles in water and ion flux across cell membranes, including cells of the inner ear and pathways of the brain used for hearing. To more fully understand the biological bases of presbycusis, 39 CBA mice, a well-studied animal model of presbycusis, underwent non-invasive hearing testing as a function of sound frequency (auditory brainstem response -- ABR thresholds, and distortion-product otoacoustic emission -- DPOAE magnitudes), and were clustered into four groups based on age and hearing ability. Aqp4 gene expression, as determined by genechip microarray analysis and quantitative real-time PCR, was compared to the young adult control group in the three older groups: middle aged with good hearing, old age with mild presbycusis, and old age with severe presbycusis. Linear regression and ANOVA showed statistically significant changes in Aqp4 gene expression and ABR and DPOAE hearing status in the cochlea and auditory midbrain -- inferior colliculus. Down-regulation in the cochlea was seen, and an initial down-, then up-regulation was discovered for the inferior colliculus Aqp4 expression. It is theorized that these changes in Aqp4 gene expression represent an age-related disruption of ion flux in the fluids of the cochlea that are responsible for ionic gradients underlying sound transduction in cochlear hair cells necessary for hearing. In regard to central auditory processing at the level of the auditory midbrain, aquaporin gene expression changes may affect neurotransmitter cycling involving supporting cells, thus impairing complex sound neural processing with age.

  12. Characterization of four hemocyanin isoforms in Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    XU Jingxiang; RUAN Lingwei; LI Zhen; YU Xiaoman; LI Sedong; SHI Hong; XU Xun

    2015-01-01

    In this study, the gene encoding hemocyanin subunit L, LvHcL, was cloned from Litopenaeus vannamei and the genomic organization was characterized. This gene was diverse with many SNPs and also had at least four isoforms, while one of them (LvHcL4) only had two exons and the exon2 was missed. Transcription analysis showed that these isoforms of LvHcL were up-regulated after WSSV challenge in WSSV-resistant shrimp, while the transcriptions were decreased constantly in WSSV-susceptible shrimp. It is suggested that the hemocyanin had rich polymorphism and was involved in the antiviral response. These results could extend our previous findings and provide insights into the immune feature of hemocyanin, which would be helpful for further studies aimed at antiviral mechanism in inver-tebrate.

  13. Aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms control lymphoid cancer cell proliferation through differentially regulating tumor suppressor p53 activity.

    Science.gov (United States)

    Gardella, Kacie A; Muro, Israel; Fang, Gloria; Sarkar, Krishnakali; Mendez, Omayra; Wright, Casey W

    2016-03-01

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is involved in xenobiotic and hypoxic responses, and we previously showed that ARNT also regulates nuclear factor-κB (NF-κB) signaling by altering the DNA binding activity of the RelB subunit. However, our initial study of ARNT-mediated RelB modulation was based on simultaneous suppression of the two ARNT isoforms, isoform 1 and 3, and precluded the examination of their individual functions. We find here that while normal lymphocytes harbor equal levels of isoform 1 and 3, lymphoid malignancies exhibit a shift to higher levels of ARNT isoform 1. These elevated levels of ARNT isoform 1 are critical to the proliferation of these cancerous cells, as suppression of isoform 1 in a human multiple myeloma (MM) cell line, and an anaplastic large cell lymphoma (ALCL) cell line, triggered S-phase cell cycle arrest, spontaneous apoptosis, and sensitized cells to doxorubicin treatment. Furthermore, co-suppression of RelB or p53 with ARNT isoform 1 prevented cell cycle arrest and blocked doxorubicin induced apoptosis. Together our findings reveal that certain blood cancers rely on ARNT isoform 1 to potentiate proliferation by antagonizing RelB and p53-dependent cell cycle arrest and apoptosis. Significantly, our results identify ARNT isoform 1 as a potential target for anticancer therapies.

  14. Regulation of NADPH oxidase 5 by protein kinase C isoforms.

    Directory of Open Access Journals (Sweden)

    Feng Chen

    Full Text Available NADPH oxidase5 (Nox5 is a novel Nox isoform which has recently been recognized as having important roles in the pathogenesis of coronary artery disease, acute myocardial infarction, fetal ventricular septal defect and cancer. The activity of Nox5 and production of reactive oxygen species is regulated by intracellular calcium levels and phosphorylation. However, the kinases that phosphorylate Nox5 remain poorly understood. Previous studies have shown that the phosphorylation of Nox5 is PKC dependent, but this contention was based on the use of pharmacological inhibitors and the isoforms of PKC involved remain unknown. Thus, the major goals of this study were to determine whether PKC can directly regulate Nox5 phosphorylation and activity, to identify which isoforms are involved in the process, and to understand the functional significance of this pathway in disease. We found that a relatively specific PKCα inhibitor, Ro-32-0432, dose-dependently inhibited PMA-induced superoxide production from Nox5. PMA-stimulated Nox5 activity was significantly reduced in cells with genetic silencing of PKCα and PKCε, enhanced by loss of PKCδ and the silencing of PKCθ expression was without effect. A constitutively active form of PKCα robustly increased basal and PMA-stimulated Nox5 activity and promoted the phosphorylation of Nox5 on Ser490, Thr494, and Ser498. In contrast, constitutively active PKCε potently inhibited both basal and PMA-dependent Nox5 activity. Co-IP and in vitro kinase assay experiments demonstrated that PKCα directly binds to Nox5 and modifies Nox5 phosphorylation and activity. Exposure of endothelial cells to high glucose significantly increased PKCα activation, and enhanced Nox5 derived superoxide in a manner that was in prevented by a PKCα inhibitor, Go 6976. In summary, our study reveals that PKCα is the primary isoform mediating the activation of Nox5 and this maybe of significance in our understanding of the vascular

  15. Protein chemical characterization of Gc globulin (vitamin D-binding protein) isoforms; Gc-1f, Gc-1s and Gc-2

    DEFF Research Database (Denmark)

    Christiansen, Maja; Jørgensen, Charlotte S; Laursen, Inga;

    2007-01-01

    exchange chromatography. The separated isoforms and several commercial preparations of individual isoforms were characterized by mass spectrometry. This revealed that the major isoforms were non-glycosylated. Compared to the Gc-1f isoform the other dominating isoforms represented an Asp/Glu substitution......Gc globulin, also called vitamin D-binding protein, is a plasma protein involved in the extracellular actin-scavenger system, vitamin D transport and possibly also other biological activities. Low levels of Gc globulin have been found to correlate with multiple organ failure and non......-survival of patients with fulminant hepatic failure and trauma. Here, we characterize the dominant isoforms of plasma-derived Gc globulin from Cohn fraction IV paste with respect to amino acid sequence and posttranslational modifications. Gc globulin was purified in large scale and the isoforms separated by ion...

  16. Profilin isoforms modulate astrocytic morphology and the motility of astrocytic processes.

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    Stefanie K Schweinhuber

    Full Text Available The morphology of astrocytic processes determines their close structural association with synapses referred to as the 'tripartite synapse'. Concerted morphological plasticity processes at tripartite synapses are supposed to shape neuronal communication. Morphological changes in astrocytes as well as the motility of astrocytic processes require remodeling of the actin cytoskeleton. Among the regulators of fast timescale actin-based motility, the actin binding protein profilin 1 has recently been shown to control the activity-dependent outgrowth of astrocytic processes. Here, we demonstrate that cultured murine astrocytes in addition to the ubiquitous profilin 1 also express the neuronal isoform profilin 2a. To analyze the cellular function of both profilins in astrocytes, we took advantage of a shRNA mediated isoform-specific downregulation. Interestingly, consistent with earlier results in neurons, we found redundant as well as isoform-specific functions of both profilins in modulating cellular physiology. The knockdown of either profilin 1 or profilin 2a led to a significant decrease in cell spreading of astrocytes. In contrast, solely the knockdown of profilin 2a resulted in a significantly reduced morphological complexity of astrocytes in both dissociated and slice culture astrocytes. Moreover, both isoforms proved to be crucial for forskolin-induced astrocytic stellation. Furthermore, forskolin treatment resulted in isoform-specific changes in the phosphorylation level of profilin 1 and profilin 2a, leading to a PKA-dependent phosphorylation of profilin 2a. In addition, transwell assays revealed an involvement of both isoforms in the motility of astrocytic processes, while FRAP analysis displayed an isoform-specific role of profilin 1 in the regulation of actin dynamics in peripheral astrocytic processes. Taken together, we suggest profilin isoforms to be important modulators of astrocytic morphology and motility with overlapping as well as

  17. Crystal Structure of an Ammonia-Permeable Aquaporin

    DEFF Research Database (Denmark)

    Kirscht, Andreas; Kaptan, Shreyas S; Bienert, Gerd Patrick;

    2016-01-01

    the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals...... an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity...

  18. Can Stabilization and Inhibition of Aquaporins Contribute to Future Development of Biomimetic Membranes?

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    Janet To

    2015-08-01

    Full Text Available In recent years, the use of biomimetic membranes that incorporate membrane proteins, i.e., biomimetic-hybrid membranes, has increased almost exponentially. Key membrane proteins in these systems have been aquaporins, which selectively permeabilize cellular membranes to water. Aquaporins may be incorporated into synthetic lipid bilayers or to more stable structures made of block copolymers or solid-state nanopores. However, translocation of aquaporins to these alien environments has adverse consequences in terms of performance and stability. Aquaporins incorporated in biomimetic membranes for use in water purification and desalination should also withstand the harsh environment that may prevail in these conditions, such as high pressure, and presence of salt or other chemicals. In this respect, modified aquaporins that can be adapted to these new environments should be developed. Another challenge is that biomimetic membranes that incorporate high densities of aquaporin should be defect-free, and this can only be efficiently ascertained with the availability of completely inactive mutants that behave otherwise like the wild type aquaporin, or with effective non-toxic water channel inhibitors that are so far inexistent. In this review, we describe approaches that can potentially be used to overcome these challenges.

  19. Regulation of the Water Channel Aquaporin-2 via 14-3-3 Theta (θ) and Zeta (ζ)

    DEFF Research Database (Denmark)

    Moeller, Hanne B; Slengerik-Hansen, Joachim; Aroankins, Takwa

    2015-01-01

    The 14-3-3 family of proteins are multifunctional proteins that interact with many of their cellular targets in a phosphorylation-dependent manner. Here, we determined that 14-3-3 proteins interact with phosphorylated forms of the water channel aquaporin-2 (AQP2) and modulate its function....... With the exception of sigma (σ), all 14-3-3 isoforms were abundantly expressed in mouse kidney and mouse kidney collecting duct cells (mpkCCD14). Long-term treatment of mpkCCD14 cells with the type 2 vasopressin receptor agonist dDAVP increased mRNA and protein levels of AQP2 alongside 14-3-3 beta (β) and zeta (ζ......256 phosphorylation critical for the interactions. shRNA-mediated knockdown of 14-3-3 ζ in mpkCCD14 cells resulted in increased AQP2 ubiquitylation, decreased AQP2 protein half-life and reduced AQP2 levels. In contrast, knockdown of 14-3-3 θ resulted in increased AQP2 half-life and increased AQP2...

  20. Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse Spatial gene

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    Mattei Marie-Geneviève

    2004-07-01

    Full Text Available Abstract Background The stromal component of the thymic microenvironment is critical for T lymphocyte generation. Thymocyte differentiation involves a cascade of coordinated stromal genes controlling thymocyte survival, lineage commitment and selection. The "Stromal Protein Associated with Thymii And Lymph-node" (Spatial gene encodes a putative transcription factor which may be involved in T-cell development. In the testis, the Spatial gene is also expressed by round spermatids during spermatogenesis. Results The Spatial gene maps to the B3-B4 region of murine chromosome 10 corresponding to the human syntenic region 10q22.1. The mouse Spatial genomic DNA is organised into 10 exons and is alternatively spliced to generate two short isoforms (Spatial-α and -γ and two other long isoforms (Spatial-δ and -ε comprising 5 additional exons on the 3' site. Here, we report the cloning of a new short isoform, Spatial-β, which differs from other isoforms by an additional alternative exon of 69 bases. This new exon encodes an interesting proline-rich signature that could confer to the 34 kDa Spatial-β protein a particular function. By quantitative TaqMan RT-PCR, we have shown that the short isoforms are highly expressed in the thymus while the long isoforms are highly expressed in the testis. We further examined the inter-species conservation of Spatial between several mammals and identified that the protein which is rich in proline and positive amino acids, is highly conserved. Conclusions The Spatial gene generates at least five alternative spliced variants: three short isoforms (Spatial-α, -β and -γ highly expressed in the thymus and two long isoforms (Spatial-δ and -ε highly expressed in the testis. These alternative spliced variants could have a tissue specific function.

  1. Preparative scale production of functional mouse aquaporin 4 using different cell-free expression modes.

    Directory of Open Access Journals (Sweden)

    Lei Kai

    Full Text Available The continuous progress in the structural and functional characterization of aquaporins increasingly attracts attention to study their roles in certain mammalian diseases. Although several structures of aquaporins have already been solved by crystallization, the challenge of producing sufficient amounts of functional proteins still remains. CF (cell free expression has emerged in recent times as a promising alternative option in order to synthesize large quantities of membrane proteins, and the focus of this report was to evaluate the potential of this technique for the production of eukaryotic aquaporins. We have selected the mouse aquaporin 4 as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based cell-free system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (cell-free membrane protein expression as precipitate mode generating initial aquaporin precipitates as well as in the D-CF (cell-free membrane protein expression in presence of detergent mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of cell-free reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated P(f value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10°C, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration.

  2. FSH isoform pattern in classic galactosemia

    OpenAIRE

    Gubbels, C.S.; Thomas, C. M.; Wodzig, K.W.H.; Olthaar, A. J.; Jaeken, J.; Sweep, F.C.; Rubio-Gozalbo, M.E.

    2010-01-01

    Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns...

  3. The time course changes in expression of aquaporin 4 and aquaporin 1 following global cerebral ischemic edema in rat

    OpenAIRE

    Gürsoy- Özdemir, Yasemin; Akdemir, G.; Kaymaz, F,; Akalan, N,; Akdemir, E.S.

    2016-01-01

    Background: The aim of this global cerebral ischemia study was to study the changes in expression levels of aquaporin 4 (AQP4) and AQP1 over time. Methods: Sprague-Dawley type male rats were divided into six groups. Sham group and ischemia/reperfusion were performed on five other groups using the four-vessel occlusion model. Reperfusion was done 30 min after the occlusion, and each group was tested at 1, 6, 12, 24, and 48 h for brain wet-dry weight ratio and AQP4 and AQP1 expression levels us...

  4. Molecular modeling study on tunnel behavior in different histone deacetylase isoforms.

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    Sundarapandian Thangapandian

    Full Text Available Histone deacetylases (HDACs have emerged as effective therapeutic targets in the treatment of various diseases including cancers as these enzymes directly involved in the epigenetic regulation of genes. However the development of isoform-selective HDAC inhibitors has been a challenge till date since all HDAC enzymes possess conserved tunnel-like active site. In this study, using molecular dynamics simulation we have analyzed the behavior of tunnels present in HDAC8, 10, and 11 enzymes of class I, II, and IV, respectively. We have identified the equivalent tunnel forming amino acids in these three isoforms and found that they are very much conserved with subtle differences to be utilized in selective inhibitor development. One amino acid, methionine of HDAC8, among six tunnel forming residues is different in isoforms of other classes (glutamic acid (E in HDAC10 and leucine (L in HDAC 11 based on which mutations were introduced in HDAC11, the less studied HDAC isoform, to observe the effects of this change. The HDAC8-like (L268M mutation in the tunnel forming residues has almost maintained the deep and narrow tunnel as present in HDAC8 whereas HDAC10-like (L268E mutation has changed the tunnel wider and shallow as observed in HDAC10. These results explained the importance of the single change in the tunnel formation in different isoforms. The observations from this study can be utilized in the development of isoform-selective HDAC inhibitors.

  5. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    Science.gov (United States)

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-01

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization.

  6. C/EBPβ Isoforms Expression in the Rat Brain during the Estrous Cycle

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    Valeria Hansberg-Pastor

    2015-01-01

    Full Text Available The CCAAT/enhancer-binding protein beta (C/EBPβ is a transcription factor expressed in different areas of the brain that regulates the expression of several genes involved in cell differentiation and proliferation. This protein has three isoforms (LAP1, LAP2, and LIP with different transcription activation potential. The role of female sex hormones in the expression pattern of C/EBPβ isoforms in the rat brain has not yet been described. In this study we demonstrate by western blot that the expression of the three C/EBPβ isoforms changes in different brain areas during the estrous cycle. In the cerebellum, LAP2 content diminished on diestrus and proestrus and LIP content diminished on proestrus and estrus days. In the prefrontal cortex, LIP content was higher on proestrus and estrus days. In the hippocampus, LAP isoforms presented a switch on diestrus day, since LAP1 content was the highest while that of LAP2 was the lowest. The LAP2 isoform was the most abundant one in all the three brain areas. The LAP/LIP ratio changed throughout the cycle and was tissue specific. These results suggest that C/EBPβ isoforms expression changes in a tissue-specific manner in the rat brain due to the changes in sex steroid hormone levels presented during the estrous cycle.

  7. Expression of mdr isoforms in mice during estrous cycle and under hormone stimulation

    Directory of Open Access Journals (Sweden)

    Marion Schiengold

    2006-01-01

    Full Text Available The multidrug resistance (MDR phenotype is associated with the expression of P-glycoprotein (Pgp, coded by the multigenic mdr family. Mice present the isoforms mdr1 and mdr3, which are responsible for multidrug resistance, and mdr2, that is involved in the transport of phospholipids. mdr1 expression has more recently been associated also with the secretion of steroid hormones. This work presents an RT-PCR analysis of the expression of mdr isoforms, in several organs of mice during different phases of the estrous cycle. Additionally, females were ovariectomized, submitted to different hormone treatments, and their uterus was analyzed for the expression of mdr isoforms. The results show that in the adrenal gland and ovaries mdr1 is the main isoform during proestrus, and that progesterone or a combination of progesterone and estrogen induce the expression of all mdr isoforms in the uterus of ovariectomized females. We suggest that the functions of mdr1 and mdr3 are overlapping, that mdr3 may be the more efficient isoform in the detoxification function, and that mdr1 may be more closely related to the secretion of steroid hormones.

  8. Glutamic acid decarboxylase isoform distribution in transgenic mouse septum: an anti-GFP immunofluorescence study.

    Science.gov (United States)

    Verimli, Ural; Sehirli, Umit S

    2016-09-01

    The septum is a basal forebrain region located between the lateral ventricles in rodents. It consists of lateral and medial divisions. Medial septal projections regulate hippocampal theta rhythm whereas lateral septal projections are involved in processes such as affective functions, memory formation, and behavioral responses. Gamma-aminobutyric acidergic neurons of the septal region possess the 65 and 67 isoforms of the enzyme glutamic acid decarboxylase. Although data on the glutamic acid decarboxylase isoform distribution in the septal region generally appears to indicate glutamic acid decarboxylase 67 dominance, different studies have given inconsistent results in this regard. The aim of this study was therefore to obtain information on the distributions of both of these glutamic acid decarboxylase isoforms in the septal region in transgenic mice. Two animal groups of glutamic acid decarboxylase-green fluorescent protein knock-in transgenic mice were utilized in the experiment. Brain sections from the region were taken for anti-green fluorescent protein immunohistochemistry in order to obtain estimated quantitative data on the number of gamma-aminobutyric acidergic neurons. Following the immunohistochemical procedures, the mean numbers of labeled cells in the lateral and medial septal nuclei were obtained for the two isoform groups. Statistical analysis yielded significant results which indicated that the 65 isoform of glutamic acid decarboxylase predominates in both lateral and medial septal nuclei (unpaired two-tailed t-test p glutamic acid decarboxylase isoform 65 in the septal region in glutamic acid decarboxylase-green fluorescent protein transgenic mice.

  9. Modulation of estrogen receptor-beta isoforms by phytoestrogens in breast cancer cells.

    Science.gov (United States)

    Cappelletti, Vera; Miodini, Patrizia; Di Fronzo, Giovanni; Daidone, Maria Grazia

    2006-05-01

    High consumption of phytoestrogen-rich food correlates with reduced incidence of breast cancer. However, the effect of phytoestrogens on growth of pre-existing breast tumors presents concerns when planning the use of phytoestrogens as chemoprevention st rategy. Genistein, the active phytoestrogen in soy, displays weak estrogenic activity mediated by estrogen receptor (ER) with a preferential binding for the ER-beta species. However, no information is at present available on the interaction between phytoestrogens and the various isoforms generated by alternative splicing. In two human breast cancer cell lines, T47D and BT20, which express variable levels of ER-beta, the effect of genistein and quercetin was evaluated singly and in comparison with 17beta-estradiol, on mRNA expression of estrogen receptor-beta (ER-beta) isoforms evaluated by a triple primer RT-PCR assay. In T47D cells estradiol caused a 6-fold up-regulation of total ER-beta, and modified the relative expression pattern of the various isoforms, up-regulating the beta2 and down-regulating the beta5 isoform. Genistein up-regulated ER-beta2 and ER-beta1 in T47D cells, and after treatment the ER-beta2 isoform became prevalent, while in BT20 cells it almost doubled the percent contribution of ER-beta1 and ER-beta2 to total ER-beta. Quercetin did not alter the total levels nor the percent distribution of ER-beta isoforms in either cell line. Genistein, through the modulation of ER-beta isoform RNA expression inhibited estrogen-promoted cell growth, without interfering on estrogen-regulated transcription. ER-beta and its ER-beta mRNA isoforms may be involved in a self-limiting mechanism of estrogenic stimulation promoted either by the natural hormone or by weaker estrogen agonists like genistein.

  10. The impact of tropomyosins on actin filament assembly is isoform specific.

    Science.gov (United States)

    Janco, Miro; Bonello, Teresa T; Byun, Alex; Coster, Adelle C F; Lebhar, Helene; Dedova, Irina; Gunning, Peter W; Böcking, Till

    2016-07-01

    Tropomyosin (Tpm) is an α helical coiled-coil dimer that forms a co-polymer along the actin filament. Tpm is involved in the regulation of actin's interaction with binding proteins as well as stabilization of the actin filament and its assembly kinetics. Recent studies show that multiple Tpm isoforms also define the functional properties of distinct actin filament populations within a cell. Subtle structural variations within well conserved Tpm isoforms are the key to their functional specificity. Therefore, we purified and characterized a comprehensive set of 8 Tpm isoforms (Tpm1.1, Tpm1.12, Tpm1.6, Tpm1.7, Tpm1.8, Tpm2.1, Tpm3.1, and Tpm4.2), using well-established actin co-sedimentation and pyrene fluorescence polymerization assays. We observed that the apparent affinity (Kd(app)) to filamentous actin varied in all Tpm isoforms between ∼0.1-5 μM with similar values for both, skeletal and cytoskeletal actin filaments. The data did not indicate any correlation between affinity and size of Tpm molecules, however high molecular weight (HMW) isoforms Tpm1.1, Tpm1.6, Tpm1.7 and Tpm2.1, showed ∼3-fold higher cooperativity compared to low molecular weight (LMW) isoforms Tpm1.12, Tpm1.8, Tpm3.1, and Tpm4.2. The rate of actin filament elongation in the presence of Tpm2.1 increased, while all other isoforms decreased the elongation rate by 27-85 %. Our study shows that the biochemical properties of Tpm isoforms are finely tuned and depend on sequence variations in alternatively spliced regions of Tpm molecules.

  11. Role of ROCK Isoforms in Regulation of Stiffness Induced Myofibroblast Differentiation in Lung Fibrosis.

    Science.gov (United States)

    Htwe, Su S; Cha, Byung H; Yue, Kan; Khademhosseini, Ali; Knox, Alan J; Ghaemmaghami, Amir M

    2017-02-22

    Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho associated coiled-coil forming kinase (ROCK) has shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in fibrotic state. There are two known ROCK isoforms in human, ROCK1 (ROKβ) and ROCK2 (ROKα), but specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a Gelatin methacryloyl (GelMA) hydrogel based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix and not soft matrix, can induce myofibroblast differentiation with high αSMA expression. Furthermore, our data confirm that the inhibition of ROCK signalling by a pharmacological inhibitor (i.e. Y27632) attenuates stiffness induced αSMA expression and fibre assembly in myofibroblasts. To assess the role of ROCK isoforms in this process we used siRNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix as opposed to soft matrix where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fibre assembly. Moreover complete loss of αSMA fibre assembly was seen only in the absence of both ROCK isoforms suggesting that both isoforms are implicated in this process. Overall our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices.

  12. Frequency and prognostic impact of antibodies to aquaporin-4 in patients with optic neuritis

    DEFF Research Database (Denmark)

    Jarius, Sven; Frederiksen, Jette Lautrup Battistini; Waters, Patrick;

    2010-01-01

    Antibodies to aquaporin-4 (AQP4-Ab) are found in 60-80% of patients with neuromyelitis optica (NMO), a severely disabling inflammatory CNS disorder of putative autoimmune aetiology, which predominantly affects the optic nerves and spinal cord....

  13. Immunodetection of aquaporin 5 in sheep salivary glands related to pasture vegetative cycle.

    Science.gov (United States)

    Scocco, Paola; Aralla, Marina; Catorci, Andrea; Belardinelli, Carlo; Arrighi, Silvana

    2011-01-01

    Mammalian aquaporins (AQPs) are a family of at least 13 integral membrane proteins expressed in various epithelia, where they function as channels to permeate water and small solutes. AQP5 is widely expressed in the exocrine gland where it is likely involved in providing an appropriate amount of fluid to be secreted with granular contents. As regards AQP5 expression in the salivary glands, literature is lacking concerning domestic animal species. This study was chiefly aimed at immunohistochemically investigating the presence and localization of AQP5 in sheep mandibular and parotid glands. In addition, AQP5 immunoreactivity was comparatively evaluated in animals fed with forage containing different amounts of water related to the pasture vegetative cycle, in order to shed light on the possible response of the gland to environmental modifications. Moderate AQP5-immunoreactivity was shown at the level of the lateral surface of mandibular serous demilune cells, not affected by the pasture vegetative cycle or water content. On the contrary, the parotid gland arcinar cells showed AQP5-immunoreactivity at the level of apical and lateral plasma membrane, which was slight to very strong, according to the pasture vegetative development and interannual climatic variations. AQP5 expression is likely due to its involvement in providing appropriate saliva fluidity. Indeed, the lowest AQP5 immunoreactivity was noticed when food water content increased.

  14. Immunodetection of aquaporin 5 in sheep salivary glands related to pasture vegetative cycle

    Directory of Open Access Journals (Sweden)

    Silvana Arrighi

    2011-10-01

    Full Text Available Mammalian aquaporins (AQPs are a family of at least 13 integral membrane proteins expressed in various epithelia, where they function as channels to permeate water and small solutes. AQP5 is widely expressed in the exocrine gland where it is likely involved in providing an appropriate amount of fluid to be secreted with granular contents. As regards AQP5 expression in the salivary glands, literature is lacking concerning domestic animal species. This study was chiefly aimed at immunohistochemically investigating the presence and localization of AQP5 in sheep mandibular and parotid glands. In addition, AQP5 immunoreactivity was comparatively evaluated in animals fed with forage containing different amounts of water related to the pasture vegetative cycle, in order to shed light on the possible response of the gland to environmental modifications. Moderate AQP5-immunoreactivity was shown at the level of the lateral surface of mandibular serous demilune cells, not affected by the pasture vegetative cycle or water content. On the contrary, the parotid gland arcinar cells showed AQP5-immunoreactivity at the level of apical and lateral plasma membrane, which was slight to very strong, according to the pasture vegetative development and interannual climatic variations. AQP5 expression is likely due to its involvement in providing appropriate saliva fluidity. Indeed, the lowest AQP5 immunoreactivity was noticed when food water content increased. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 3, pp. 458–464

  15. Frequency and prognostic impact of antibodies to aquaporin-4 in patients with optic neuritis

    DEFF Research Database (Denmark)

    Jarius, Sven; Frederiksen, Jette Lautrup Battistini; Waters, Patrick;

    2010-01-01

    Antibodies to aquaporin-4 (AQP4-Ab) are found in 60-80% of patients with neuromyelitis optica (NMO), a severely disabling inflammatory CNS disorder of putative autoimmune aetiology, which predominantly affects the optic nerves and spinal cord.......Antibodies to aquaporin-4 (AQP4-Ab) are found in 60-80% of patients with neuromyelitis optica (NMO), a severely disabling inflammatory CNS disorder of putative autoimmune aetiology, which predominantly affects the optic nerves and spinal cord....

  16. Antiangiogenic VEGF Isoform in Inflammatory Myopathies

    Directory of Open Access Journals (Sweden)

    Nila Volpi

    2013-01-01

    Full Text Available Objective. To investigate expression of vascular endothelial growth factor (VEGF antiangiogenic isoform A-165b on human muscle in idiopathic inflammatory myopathies (IIM and to compare distribution of angiogenic/antiangiogenic VEGFs, as isoforms shifts are described in other autoimmune disorders. Subjects and Methods. We analyzed VEGF-A165b and VEGF-A by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with IIM (polymyositis, dermatomyositis, and inclusion body myositis and 6 control muscle samples. TGF-β, a prominent VEGF inductor, was analogously evaluated. Intergroup differences of western blot bands density were statistically examined. Endomysial vascularization, inflammatory score, and muscle regeneration, as pathological parameters of IIM, were quantitatively determined and their levels were confronted with VEGF expression. Results. VEGF-A165b was significantly upregulated in IIM, as well as TGF-β. VEGF-A was diffusely expressed on unaffected myofibers, whereas regenerating/atrophic myofibres strongly reacted for both VEGF-A isoforms. Most inflammatory cells and endomysial vessels expressed both isoforms. VEGF-A165b levels were in positive correlation to inflammatory score, endomysial vascularization, and TGF-β. Conclusions. Our findings indicate skeletal muscle expression of antiangiogenic VEGF-A165b and preferential upregulation in IIM, suggesting that modulation of VEGF-A isoforms may occur in myositides.

  17. Expression of 14-3-3 protein isoforms in mouse oocytes, eggs and ovarian follicular development

    Directory of Open Access Journals (Sweden)

    De Santanu

    2012-01-01

    Full Text Available Abstract Background The 14-3-3 (YWHA proteins are a highly conserved, ubiquitously expressed family of proteins. Seven mammalian isoforms of 14-3-3 are known (β, γ, ε, ζ, η, τ and, σ. These proteins associate with many intracellular proteins involved in a variety of cellular processes including regulation of the cell cycle, metabolism and protein trafficking. We are particularly interested in the role of 14-3-3 in meiosis in mammalian eggs and the role 14-3-3 proteins may play in ovarian function. Therefore, we examined the expression of 14-3-3 proteins in mouse oocyte and egg extracts by Western blotting after polyacrylamide gel electrophoresis, viewed fixed cells by indirect immunofluorescence, and examined mouse ovarian cells by immunohistochemical staining to study the expression of the different 14-3-3 isoforms. Results We have determined that all of the mammalian 14-3-3 isoforms are expressed in mouse eggs and ovarian follicular cells including oocytes. Immunofluorescence confocal microscopy of isolated oocytes and eggs confirmed the presence of all of the isoforms with characteristic differences in some of their intracellular localizations. For example, some isoforms (β, ε, γ, and ζ are expressed more prominently in peripheral cytoplasm compared to the germinal vesicles in oocytes, but are uniformly dispersed within eggs. On the other hand, 14-3-3η is diffusely dispersed in the oocyte, but attains a uniform punctate distribution in the egg with marked accumulation in the region of the meiotic spindle apparatus. Immunohistochemical staining detected all isoforms within ovarian follicles, with some similarities as well as notable differences in relative amounts, localizations and patterns of expression in multiple cell types at various stages of follicular development. Conclusions We found that mouse oocytes, eggs and follicular cells within the ovary express all seven isoforms of the 14-3-3 protein. Examination of the

  18. Roles of different IRES-dependent FGF2 isoforms in the acquisition of the major aggressive features of human metastatic melanoma.

    Science.gov (United States)

    Andreucci, Elena; Bianchini, Francesca; Biagioni, Alessio; Del Rosso, Mario; Papucci, Laura; Schiavone, Nicola; Magnelli, Lucia

    2017-01-01

    Fibroblast growth factor 2 (FGF2) is involved in many physiological and pathological processes. Fgf2 deregulation contributes to the acquisition of malignant features of melanoma and other cancers. FGF2 is an alternative translation product expressed as five isoforms, a low-molecular-weight (18 KDa) and four high-molecular-weight (22, 22.5, 24, 34 KDa) isoforms, with different subcellular distributions. An internal ribosomal entry site (IRES) in its mRNA controls the translation of all the isoforms with the exception for the cap-dependent 34 KDa. The 18-KDa isoform has been extensively studied, while very few is known about the roles of high molecular weight isoforms. FGF2 is known to promote melanoma development and progression. To disclose the differential contribution of FGF2 isoforms in melanoma, we forced the expression of IRES-dependent low-molecular-weight (LMW, 18 KDa) and high-molecular-weight (HMW, 22, 22.5, 24 KDa) isoforms in a human metastatic melanoma cell line. This comparative study highlights that, while LMW isoform confers stem-like features to melanoma cells and promotes angiogenesis, HMW isoforms induce higher migratory ability and contribute to tumor perfusion by promoting vasculogenic mimicry (VM) when endothelial cell-driven angiogenesis is lacking. To conclude, FGF2 isoforms mainly behave in specific, antithetical manners, but can cooperate in different steps of tumor progression, providing melanoma cells with major malignant features.

  19. Regulation of the water channel aquaporin-2 by posttranslational modification.

    Science.gov (United States)

    Moeller, Hanne B; Olesen, Emma T B; Fenton, Robert A

    2011-05-01

    The cellular functions of many eukaryotic membrane proteins, including the vasopressin-regulated water channel aquaporin-2 (AQP2), are regulated by posttranslational modifications. In this article, we discuss the experimental discoveries that have advanced our understanding of how posttranslational modifications affect AQP2 function, especially as they relate to the role of AQP2 in the kidney. We review the most recent data demonstrating that glycosylation and, in particular, phosphorylation and ubiquitination are mechanisms that regulate AQP2 activity, subcellular sorting and distribution, degradation, and protein interactions. From a clinical perspective, posttranslational modification resulting in protein misrouting or degradation may explain certain forms of nephrogenic diabetes insipidus. In addition to providing major insight into the function and dynamics of renal AQP2 regulation, the analysis of AQP2 posttranslational modification may provide general clues as to the role of posttranslational modification for regulation of other membrane proteins.

  20. Aquaporin-1 is a Maxwell's Demon in the Body

    CERN Document Server

    Shu, Liangsuo; Xiaokang,; Qian, Liu Xin; Huang, Suyi; Jin, Shiping; Yang, Baoxue

    2015-01-01

    Aquaporin-1 (AQP1) is a membrane protein which is selectively permeable to water. Due to its hourglass shape, AQP1 can sense the information of solute molecules in osmosis. At the cost of consuming this information, AQP1 can move water against its chemical potential gradient: it works as one kind of Maxwell's Demon. This effect was detected quantitatively by measuring the water osmosis of mice erythrocytes. This ability may protect the erythrocytes from the eryptosis elicited by osmotic shock when they move in the kidney, where a large gradient of urea is required for the urine concentrating mechanism. This finding anticipates a new beginning of inquiries into the complicated relationships among mass, energy and information in bio-systems.

  1. Acetazolamide inhibits aquaporin-1 protein expression and angiogenesis

    Institute of Scientific and Technical Information of China (English)

    Yang XIANG; Bing MA; Tao LI; Jun-wei GAO; He-ming YU; Xue-jun LI

    2004-01-01

    AIM: To study effects of acetazolamide on aquaporin-1 (AQP1) protein expression and angiogenesis. METHODS:Establishing Lewis-lung-carcinoma model, the localization of AQP1 in tumor tissues was investigated by immunohistochemical methods; The biological activity of acetazolamide was detected by endothelial cells proliferation test (MTT) assay and chorioallantoic membrane (CAM) vascular inhibition test. RESULTS: Immunohistochemical localization of AQP1 in mice tumor was labeled in capillaries, post capillary venules endothelial cells. After being treated with acetazolamide, the number of capillaries and post capillary venules was significantly decreased in tumor tissue. Acetazolamide showed significant inhibitory effect on angiogenesis in CAM and endothelial cell proliferation.CONCLUSION: Acetazolamide might be identified and developed as one of potential lead compounds for a new therapeutic intervention in inhibiting cancer angiogenesis.

  2. Aquaporins 6-12 in the human eye

    DEFF Research Database (Denmark)

    Tran, Thuy Linh; Bek, Toke; Holm, Lars;

    2013-01-01

    Purpose: Aquaporins (AQPs) are widely expressed and have diverse distribution patterns in the eye. AQPs 0-5 have been localized at the cellular level in human eyes. We investigated the presence of the more recently discovered AQPs 6-12 in the human eye. Methods: RT-PCR was performed on fresh tissue...... from two human eyes divided into the cornea, corneal limbus, ciliary body and iris, lens, choroid, optic nerve, retina and sclera. Each structure was examined to detect the mRNA of AQPs 6-12. Twenty-one human eyes were examined using immunohistochemical and immunofluorescence techniques to determine...... in the nonpigmented ciliary epithelium and retinal ganglion cells. AQP11 immunolabelling was detected in the corneo-limbal epithelium, nonpigmented ciliary epithelium and inner limiting membrane of the retina. Conclusion: Selective expression of AQP7, AQP9 and AQP11 was found within various structures of the human...

  3. Effect of aquaporin-q deletion on pleural fluid transport

    Institute of Scientific and Technical Information of China (English)

    JIANGJin-Jun; HONGQun-Ying; 等

    2003-01-01

    AIM:To investigate the role of aquaporin-1(AQP1)and sodium channel on pleural fluid transport.METHODS:Wild-type and AQP1 null mice were used in this study.After the mice were briefly anesthetized,0.25mL of hyperosmolar or isosmolar solution(containing terbutaline,amiloride or saline only)was infused into the pleural space.Then mice were sacrificed at scheduled times for measurement of pleural fluid osmolality or volume,RESULTS:After instillation of hyperosmolar fluid into the pleural space,the osmolality of pleural fluid in wild-type mice was higher than that in AQP1 null mice killed at the same time(1,2,5min).There was no difference in the isosmolar clearance between the wild-type and AQP1 null mice after injection of 0.25mL isosmolar fluid into the pleural space.Terbutaline increased the osmotic and isosmolar fluid transport across pleura,but these effects were not influenced by AQP1 dfeletion.In contrast,amiloride reduced osmotic and isosmolar pleural fluid transport and these effects were not influenced by AQP1 deletion.CONCLUSION;AQP1 water channels facilitated osmotic fluid transport across the pleural surface,However,AQP1 did not play an important role in pleural isosmolar fluid clearance.Sodium channel may play a role in osmotic and isosmolar pleural fluid transport.The effects of sodium channel on fluid transport across pleural space were not influenced by aquaporin-1 deletion.

  4. Ikaros isoforms:The saga continues

    Institute of Scientific and Technical Information of China (English)

    Laura; A; Perez-Casellas; Aleksandar; Savic; Sinisa; Dovat

    2011-01-01

    Through alternate splicing,the Ikaros gene produces multiple proteins.Ikaros is essential for normal hematopoiesis and possesses tumor suppressor activity.Ikaros isoforms interact to form dimers and potentially multimeric complexes.Diverse Ikaros complexes produced by the presence of different Ikaros isoforms are hypothesized to confer distinct functions.Small dominantnegative Ikaros isoforms have been shown to inhibit the tumor suppressor activity of full-length Ikaros.Here,we describe how Ikaros activity is regulated by the coordinated expression of the largest Ikaros isoforms IK-1 and IK-H.Although IK-1 is described as full-length Ikaros,IK-H is the longest Ikaros isoform.IK-H,which includes residues coded by exon 3B (60 bp that lie between exons 3 and 4),is abundant in human but not murine hematopoietic cells.Specific residues that lie within the 20 amino acids encoded by exon 3B give IK-H DNA-binding characteristics that are distinct from those of IK-1.Moreover,IK-H can potentiate or inhibit the ability of IK-1 to bind DNA.IK-H binds to the regulatory regions of genes that are upregulated by Ikaros,but not genes that are repressed by Ikaros.Although IK-1 localizes to pericentromeric heterochromatin,IK-H can be found in both pericentromeric and non-pericentromeric locations.Anti-silencing activity of gamma satellite DNA has been shown to depend on the binding of IK-H,but not other Ikaros isoforms.The unique features of IK-H,its influence on Ikaros activity,and the lack of IK-H expression in mice suggest that Ikaros function in humans may be more complex and possibly distinct from that in mice.

  5. Comment on: Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in the gastrointestinal system

    Institute of Scientific and Technical Information of China (English)

    Ali Mobasheri

    2006-01-01

    @@ TO THE EDITOR Sir, I read with great interest the recently published article in the World Journal of Gastroenterology by Jin and co-workers[1] on the cloning and characterization of porcine aquaporin 1 water channel from the pig liver and studies on its expression in the porcine gastrointestinal system. The authors should be congratulated for making this important and valuable contribution to the field of aquaporin biology and porcine gastrointestinal physiology.However, there are a number of unresolved issues and controversies concerning the expression of aquaporins (especially aquaporin 1) in the gastrointestinal system that are worthy of additional comment and discussion by Jin and co-workers.

  6. Cloning, Characterization, and Expression Patterns of Three Sarco/Endoplasmic Reticulum Ca2+-ATPase Isoforms from Pearl Oyster (Pinctada fucata)

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A large amount of calcium is required for mollusk biomineralization. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is a well-known protein with the function of sustaining the calcium homeostasis. How does it possibly function in the process of pearl oyster biomineralization? Three SERCA isoforms, namely PSERA, PSERB, and PSERC were cloned from the pearl oyster, Pinctada fucata. The cDNAs of the three isoforms were isolated by reverse transcription-polymerase chain reaction (RT-PCR)and rapid amplification of cDNA ends. PSERA consisted of 3568 bp encoding 1007 amino acids, PSERB included 3953 bp encoding 1024 amino acids, and PSERC comprised of 3450 bp encoding 1000 amino acids.The three isoforms showed high homology (65%-87%) with SERCAs from other species. Consistent with the results from other invertebrates, Southern blot analysis revealed that the three isoforms originated from a single gene that was also related to SERCA1, SERCA2, and SERCA3 of vertebrates. The splicing mechanism of the three isoforms was similar to that of isoforms of vertebrate SERCA3. Semiquantitative RT-PCR was carried out to study the expression patterns of the three isoforms. The results showed that PSERB was ubiquitously expressed in all tested tissues and was a potential "housekeeping" SERCA isoform; PSERA was expressed in the adductor muscle and foot and was likely to be a muscle-specific isoform, and PSERC was expressed in the other tissues except the adductor muscle or foot with the highest expression levels in the gill and mantle, indicating that it was a non-muscle-specific isoform and might be involved in calcium homeostasis during pearl oyster biomineralization.

  7. Aquaporin and ocular diseases%水通道蛋白与眼科疾病

    Institute of Scientific and Technical Information of China (English)

    宋雨晨; 姚进; 蒋沁

    2015-01-01

    Aquaporin (AQP) is a type of water transporting protein expressed mainly in epithelia and endothelia of human and other mammalian species.AQP,which plays an important role in ocular function,such as maintaining normal introcular pressure(IOP) and facilitating cornea epithelium restore after injure,is abundant in ocular tissue and involved in ocular pathological processes.For example,AQP0,which was expressed in lens,its gene mutation would cause congenital cataract and abnormality in quantity or distribution would accelerate the development of age-related cataract.Besides that,dysregulation of AQP expression might cause numerous ocular disorders which related to water transport,such as macular edema of diabetic retinopathy (DR),high IOP of glaucoma,uveitis and optica neuritis.A safe and effective approach interfering the quantity and distribution of AQP expression within ocular tissues might represent a potential target for future therapeutic development of ocular diseases associated with fluid transporting disorder.%水通道蛋白(aquaporin,AQP)是一类广泛存在于哺乳动物上皮和内皮细胞膜上的特异性转运水蛋白,在眼部组织中大量存在,并参与包括维持眼压、促进上皮修复等在内的一系列病理生理过程.近年研究发现,其与眼科疾病的发生发展也密不可分,AQP0广泛大量存在于晶状体中,其基因突变导致先天性白内障发生,其后天表达改变会导致年龄相关性白内障的加速发展;眼内介导水分运输的AQP表达失调,会导致糖尿病视网膜病变的黄斑水肿、青光眼的高眼压及葡萄膜炎、视神经炎等一系列与水分子运输失衡相关的眼病.如果在眼内可以使用安全有效的技术手段从数量及空间分布上改变AQP的表达,这很可能成为治疗眼科疾病的一个新靶点.

  8. Crystallization and identification of the glycosylated moieties of two isoforms of the main allergen Hev b 2 and preliminary X-ray analysis of two polymorphs of isoform II

    Energy Technology Data Exchange (ETDEWEB)

    Fuentes-Silva, D. [Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Mendoza-Hernández, G. [Facultad de Medicina, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Stojanoff, V. [Brookhaven National Laboratory, National Synchrotron Light Source, Upton, NY (United States); Palomares, L. A. [Instituto de Biotecnología, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Zenteno, E. [Facultad de Medicina, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Torres-Larios, A. [Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico); Rodríguez-Romero, A., E-mail: adela@servidor.unam.mx [Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Cuidad Universitaria, Coyoacán, México, DF 04510 (Mexico)

    2007-09-01

    Crystallization of important glycoenzymes involved in IgE-mediated latex allergy. Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a β-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content constisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapour-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4{sub 1} with unit-cell parameters a = b = 150.17, c = 77.41 Å were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 Å, β = 113.6°. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.

  9. Recombinant Production of Human Aquaporin-1 to an Exceptional High Membrane Density in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bomholt, Julie; Helix Nielsen, Claus; Scharff-Poulsen, Peter;

    2013-01-01

    In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C-terminally tag......In the present paper we explored the capacity of yeast Saccharomyces cerevisiae as host for heterologous expression of human Aquaporin-1. Aquaporin-1 cDNA was expressed from a galactose inducible promoter situated on a plasmid with an adjustable copy number. Human Aquaporin-1 was C...... and generated a monodisperse protein preparation. A single Ni-affinity chromatography step was used to obtain almost pure Aquaporin-1. Recombinant Aquaporin-1 produced in S. cerevisiae was not N-glycosylated in contrast to the protein found in human erythrocytes....

  10. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines

    Directory of Open Access Journals (Sweden)

    Cui Zhu

    2016-08-01

    Full Text Available Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1–11 have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes, goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

  11. Urinary Excretion of Kidney Aquaporins as Possible Diagnostic Biomarker of Diabetic Nephropathy

    Science.gov (United States)

    Rossi, Luigi; Nicoletti, Maria Celeste; Mastrofrancesco, Lisa; Di Franco, Antonella; Indrio, Francesca; Lella, Rossella; Laviola, Luigi; Giorgino, Francesco; Svelto, Maria; Gesualdo, Loreto

    2017-01-01

    Diabetic nephropathy (DN) is a microangiopathic complication of diabetes mellitus (DM) affecting one-third of diabetic patients. The large variability in the clinical presentation of renal involvement in patients with DM makes kidney biopsy a prerequisite for a correct diagnosis. However, renal biopsy is an invasive procedure associated with risk of major complications. Numerous studies aimed to identify a noninvasive biomarker of DN but, so far, none of these is considered to be sufficiently specific and sensitive. Water channel aquaporins (AQPs), expressed at the plasma membrane of epithelial tubular cells, are often dysregulated during DN. In this work, we analyzed the urine excretion of AQP5 and AQP2 (uAQP5 and uAQP2), via exosomes, in 35 diabetic patients: 12 normoalbuminuric with normal renal function (DM), 11 with proteinuric nondiabetic nephropathy (NDN), and 12 with histological diagnosis and classification of DN. ELISA and WB analysis independently showed that uAQP5 was significantly increased in DN patients. Interestingly, linear regression analysis showed a positive correlation between uAQP5 and the histological class of DN. The same analysis, focusing on uAQP2, showed comparable results. Taken together, these data suggest a possible use of AQP5 and AQP2 as novel noninvasive biomarkers to help in classifying the clinical stage of DN. PMID:28246612

  12. Two TIP-like genes encoding aquaporins are expressed in sunflower guard cells.

    Science.gov (United States)

    Sarda, X; Tousch, D; Ferrare, K; Legrand, E; Dupuis, J M; Casse-Delbart, F; Lamaze, T

    1997-11-01

    SunTIP7 and SunTIP20 are closely related sunflower cDNAs showing a deduced amino acid sequence homologous to proteins of the tonoplast intrinsic protein (TIP) family. Their expression in Xenopus oocytes caused a marked increase in osmotic water permeability (demonstrating that they are water channels) which was sensitive to mercury. In leaves, in situ hybridization revealed that both SunTIP7 and SunTIP20 mRNA accumulated in the guard cells. The possible involvement of SunTIPs in stomatal movement was examined by comparing the time course of transcript accumulation and leaf conductance during the daily cycle and following a water limitation. SunTIP7 mRNA fluctuations fitted changes occurring in leaf conductance. The transcript levels were markedly and systematically increased during stomatal closure. It is suggested that aquaporin SunTIP7 facilitates water exit associated with a decrease in guard cell volume. In the same conditions, the transcript level of SunTIP20 remained constant indicating that SunTIP genes are differentially regulated within the same cell.

  13. Urinary Excretion of Kidney Aquaporins as Possible Diagnostic Biomarker of Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Luigi Rossi

    2017-01-01

    Full Text Available Diabetic nephropathy (DN is a microangiopathic complication of diabetes mellitus (DM affecting one-third of diabetic patients. The large variability in the clinical presentation of renal involvement in patients with DM makes kidney biopsy a prerequisite for a correct diagnosis. However, renal biopsy is an invasive procedure associated with risk of major complications. Numerous studies aimed to identify a noninvasive biomarker of DN but, so far, none of these is considered to be sufficiently specific and sensitive. Water channel aquaporins (AQPs, expressed at the plasma membrane of epithelial tubular cells, are often dysregulated during DN. In this work, we analyzed the urine excretion of AQP5 and AQP2 (uAQP5 and uAQP2, via exosomes, in 35 diabetic patients: 12 normoalbuminuric with normal renal function (DM, 11 with proteinuric nondiabetic nephropathy (NDN, and 12 with histological diagnosis and classification of DN. ELISA and WB analysis independently showed that uAQP5 was significantly increased in DN patients. Interestingly, linear regression analysis showed a positive correlation between uAQP5 and the histological class of DN. The same analysis, focusing on uAQP2, showed comparable results. Taken together, these data suggest a possible use of AQP5 and AQP2 as novel noninvasive biomarkers to help in classifying the clinical stage of DN.

  14. Genome-wide identification and characterization of aquaporin gene family in moso bamboo (Phyllostachys edulis).

    Science.gov (United States)

    Sun, Huayu; Li, Lichao; Lou, Yongfeng; Zhao, Hansheng; Gao, Zhimin

    2016-05-01

    Aquaporins (AQPs) are known to play a major role in maintaining water and hydraulic conductivity balance in the plant system. Numerous studies have showed AQPs execute multi-function throughout plant growth and development, including water transport, nitrogen, carbon, and micronutrient acquisition etc. However, little information on AQPs is known in bamboo. In this study, we present the first genome-wide identification and characterization of AQP genes in moso bamboo (Phyllostachys edulis) using bioinformatics. In total, 26 AQP genes were identified by homologous analysis, which were divided into four groups (PIPs, TIPs, NIPs, and SIPs) based on the phylogenetic analysis. All the genes were located on 26 different scaffolds respectively on basis of the gene mapped to bamboo genome. Evolutionary analysis indicated that Ph. edulis was more close to Oryza sativa than Zea mays in the genetic relationship. Besides, qRT-PCR was used to analyze gene expression profiles, which revealed that AQP genes were expressed constitutively in all the detected tissues, and were all responsive to the environmental cues such as drought, water, and NaCl stresses. This data suggested that AQPs may play fundamental roles in maintaining normal growth and development of bamboo, which would contribute to better understanding for the complex regulation mechanism involved in the fast-growing process of bamboo. Furthermore, the result could provide valuable information for further research on bamboo functional genomics.

  15. The Effects of Aquaporin-1 in Pulmonary Edema Induced by Fat Embolism Syndrome.

    Science.gov (United States)

    Zhang, Yiwei; Tian, Kun; Wang, Yan; Zhang, Rong; Shang, Jiawei; Jiang, Wei; Wang, Aizhong

    2016-07-21

    This study was designed to investigate the role of aquaporin1 (AQP1) in the pathologic process of pulmonary edema induced by fat embolism syndrome (FES) and the effects of a free fatty acid (FFA) mixture on AQP1 expression in pulmonary microvascular endothelial cells (PMVECs). In vivo, edema was more serious in FES mice compared with the control group. The expression of AQP1 and the wet-to-dry lung weight ratio (W/D) in the FES group were significantly increased compared with the control group. At the same time, inhibition of AQP1 decreased the pathological damage resulting from pulmonary edema. Then we performed a study in vitro to investigate whether AQP1 was induced by FFA release in FES. The mRNA and protein level of AQP1 were increased by FFAs in a dose- and time-dependent manner in PMVECs. In addition, the up-regulation of AQP1 was blocked by the inhibitor of p38 kinase, implicating the p38 MAPK pathway as involved in the FFA-induced AQP1 up-regulation in PMVECs. Our results demonstrate that AQP1 may play important roles in pulmonary edema induced by FES and can be regarded as a new therapy target for treatment of pulmonary edema induced by FES.

  16. Expression and localization of aquaporin 1b during oocyte development in the Japanese eel (Anguilla japonica

    Directory of Open Access Journals (Sweden)

    Matsubara Hajime

    2011-05-01

    Full Text Available Abstract To elucidate the molecular mechanisms underling hydration during oocyte maturation, we characterized the structure of Japanese eel (Anguilla japonica novel-water selective aquaporin 1 (AQP1b that thought to be involved in oocyte hydration. The aqp1b cDNA encodes a 263 amino acid protein that includes the six potential transmembrane domains and two Asn-Pro-Ala motifs. Reverse transcription-polymerase chain reaction showed transcription of Japanese eel aqp1b in ovary and testis but not in the other tissues. In situ hybridization studies with the eel aqp1b cRNA probe revealed intense eel aqp1b signal in the oocytes at the perinucleolus stage and the signals became faint during the process of oocyte development. Light microscopic immunocytochemical analysis of ovary revealed that the Japanese eel AQP1b was expressed in the cytoplasm around the yolk globules which were located in the peripheral region of oocytes during the primary yolk globule stage; thereafter, the immunoreactivity was observed throughout the cytoplasm of oocyte as vitellogenesis progressed. The immunoreactivity became localized around the large membrane-limited yolk masses which were formed by the fusion of yolk globules during the oocyte maturation phase. These results together indicate that AQP1b, which is synthesized in the oocyte during the process of oocyte growth, is essential for mediating water uptake into eel oocytes.

  17. Selective regulation of maize plasma membrane aquaporin trafficking and activity by the SNARE SYP121.

    Science.gov (United States)

    Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R; Chaumont, François

    2012-08-01

    Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K(+) channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K(+) channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.

  18. Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121[W

    Science.gov (United States)

    Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S.; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R.; Chaumont, François

    2012-01-01

    Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K+ channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K+ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis. PMID:22942383

  19. Overexpression of the wheat aquaporin gene, TaAQP7, enhances drought tolerance in transgenic tobacco.

    Directory of Open Access Journals (Sweden)

    Shiyi Zhou

    Full Text Available Aquaporin (AQP proteins have been shown to transport water and other small molecules through biological membranes, which is crucial for plants to combat stress caused by drought. However, the precise role of AQPs in drought stress response is not completely understood in plants. In this study, a PIP2 subgroup gene AQP, designated as TaAQP7, was cloned and characterized from wheat. Expression of TaAQP7-GFP fusion protein revealed its localization in the plasma membrane. TaAQP7 exhibited high water channel activity in Xenopus laevis oocytes and TaAQP7 transcript was induced by dehydration, and treatments with polyethylene glycol (PEG, abscisic acid (ABA and H(2O(2. Further, TaAQP7 was upregulated after PEG treatment and was blocked by inhibitors of ABA biosynthesis, implying that ABA signaling was involved in the upregulation of TaAQP7 after PEG treatment. Overexpression of TaAQP7 increased drought tolerance in tobacco. The transgenic tobacco lines had lower levels of malondialdehyde (MDA and H(2O(2, and less ion leakage (IL, but higher relative water content (RWC and superoxide dismutase (SOD and catalase (CAT activities when compared with the wild type (WT under drought stress. Taken together, our results show that TaAQP7 confers drought stress tolerance in transgenic tobacco by increasing the ability to retain water, reduce ROS accumulation and membrane damage, and enhance the activities of antioxidants.

  20. Aquaporin 4 in the sensory organs of adult zebrafish (Danio rerio).

    Science.gov (United States)

    Zichichi, Rosalia; Magnoli, Domenico; Montalbano, Giuseppe; Laurà, Rosaria; Vega, José A; Ciriaco, Emilia; Germanà, Antonino

    2011-04-12

    The aquaporins (AQPs) are a family (AQP-AQP10) of transmembrane channel proteins that mediate the transport of water, ions, gases, and small molecules across the cell membrane, thus regulating cell homeostasis. AQP4 has the highest water permeability and it is involved in hearing and vision in mammals. Here, we used immunohistochemistry to map the presence of AQP4 in the sensory organs of adult zebrafish. The antibody used detected by Western blot proteins of 34 kDa (equivalent to that of mammalian AQP4) and maps in the sensory cells of taste buds, the hair sensory cells of the neuromast and of the maculae, and cristae ampullaris of the inner ear. Moreover, the retinal photoreceptors display AQP4 immunoreactivity. The non-sensory cells in these organs were AQP4 negative. These results suggest that the AQP4 could play a role in the regulation of water balance and ion transport in the sensory cells of zebrafish, bringing new data for the utilizing of this experimental model in the biology of sensory system.

  1. Loss of aquaporin-4 expression and putative function in non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Schnabel Philipp A

    2011-05-01

    Full Text Available Abstract Background Aquaporins (AQPs have been recognized to promote tumor progression, invasion, and metastasis and are therefore recognized as promising targets for novel anti-cancer therapies. Potentially relevant AQPs in distinct cancer entities can be determined by a comprehensive expression analysis of the 13 human AQPs. Methods We analyzed the presence of all AQP transcripts in 576 different normal lung and non-small cell lung cancer (NSCLC samples using microarray data and validated our findings by qRT-PCR and immunohistochemistry. Results Variable expression of several AQPs (AQP1, -3, -4, and -5 was found in NSCLC and normal lung tissues. Furthermore, we identified remarkable differences between NSCLC subtypes in regard to AQP1, -3 and -4 expression. Higher transcript and protein levels of AQP4 in well-differentiated lung adenocarcinomas suggested an association with a more favourable prognosis. Beyond water transport, data mining of co-expressed genes indicated an involvement of AQP4 in cell-cell signalling, cellular movement and lipid metabolism, and underlined the association of AQP4 to important physiological functions in benign lung tissue. Conclusions Our findings accentuate the need to identify functional differences and redundancies of active AQPs in normal and tumor cells in order to assess their value as promising drug targets.

  2. The enhanced drought tolerance of rice plants under ammonium is related to aquaporin (AQP).

    Science.gov (United States)

    Ding, Lei; Gao, Cuimin; Li, Yingrui; Li, Yong; Zhu, Yiyong; Xu, Guohua; Shen, Qirong; Kaldenhoff, Ralf; Kai, Lei; Guo, Shiwei

    2015-05-01

    Previously, we demonstrated that drought resistance in rice seedlings was increased by ammonium (NH4(+)) treatment, but not by nitrate (NO3(-)) treatment, and that the change was associated with root development. To study the effects of different forms of nitrogen on water uptake and root growth under drought conditions, we subjected two rice cultivars (cv. 'Shanyou 63' hybrid indica and cv. 'Yangdao 6' indica, China) to polyethylene glycol-induced drought stress in a glasshouse using hydroponic culture. Under drought conditions, NH4(+) significantly stimulated root growth compared to NO3(-), as indicated by the root length, surface area, volume, and numbers of lateral roots and root tips. Drought stress decreased the root elongation rate in both cultivars when they were supplied with NO3(-), while the rate was unaffected in the presence of NH4(+). Drought stress significantly increased root protoplast water permeability, root hydraulic conductivity, and the expression of root aquaporin (AQP) plasma intrinsic protein (PIP) genes in rice plants supplied with NH4(+); these changes were not observed in plants supplied with NO3(-). Additionally, ethylene, which is involved in the regulation of root growth, accumulated in rice roots supplied with NO3(-) under conditions of drought stress. We conclude that the increase in AQP expression and/or activity enhanced the root water uptake ability and the drought tolerance of rice plants supplied with NH4(+).

  3. Hyperglycemia Induces Cellular Hypoxia through Production of Mitochondrial ROS Followed by Suppression of Aquaporin-1.

    Directory of Open Access Journals (Sweden)

    Kiminori Sada

    Full Text Available We previously proposed that hyperglycemia-induced mitochondrial reactive oxygen species (mtROS generation is a key event in the development of diabetic complications. Interestingly, some common aspects exist between hyperglycemia and hypoxia-induced phenomena. Thus, hyperglycemia may induce cellular hypoxia, and this phenomenon may also be involved in the pathogenesis of diabetic complications. In endothelial cells (ECs, cellular hypoxia increased after incubation with high glucose (HG. A similar phenomenon was observed in glomeruli of diabetic mice. HG-induced cellular hypoxia was suppressed by mitochondria blockades or manganese superoxide dismutase (MnSOD overexpression, which is a specific SOD for mtROS. Overexpression of MnSOD also increased the expression of aquaporin-1 (AQP1, a water and oxygen channel. AQP1 overexpression in ECs suppressed hyperglycemia-induced cellular hypoxia, endothelin-1 and fibronectin overproduction, and apoptosis. Therefore, hyperglycemia-induced cellular hypoxia and mtROS generation may promote hyperglycemic damage in a coordinated manner.

  4. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines.

    Science.gov (United States)

    Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

    2016-08-29

    Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1-11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

  5. Aquaporin 3 and 8 are down-regulated in TNBS-induced rat colitis.

    Science.gov (United States)

    Zhao, Guangxi; Li, Jing; Wang, Jiyao; Shen, Xizhong; Sun, Jianyong

    2014-01-03

    Aquaporins (AQPs) plays an important role in transcellular water movement, but the AQPs expression profile has not been demonstrated in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis which closely mimics human Crohn's disease (CD) histopathologically. To solve the problem, 30 female Sprague-Dawley (SD) rats were randomly divided into a model group (n=18), an ethanol control group (n=6) and a normal control group (n=6). On day 1, the rats in the model group received TNBS+50% ethanol via the rectum, while the ethanol control rats received an equal volume of 50% ethanol and the normal control rats did not receive any treatment. All rats were sacrificed on day 7, and ileum, proximal colon and distal colon specimens were obtained to examine the alteration in AQP3 and AQP8 using real-time polymerase chain reaction, Western blot analysis and immunohistochemistry. As a result, exposure to TNBS+ethanol resulted in a marked decrease in both the mRNA and protein expression of AQP3 and AQP8, with the exception of AQP8 protein which was negative in the distal colon in all three groups. These reductions in AQP3 and AQP8 were accompanied by an increase in intestinal inflammation and injury. The results obtained here implied that both AQP3 and AQP8 may be involved in the pathogenesis of inflammatory bowel disease.

  6. Isoforms of receptors of fibroblast growth factors.

    Science.gov (United States)

    Gong, Siew-Ging

    2014-12-01

    The breadth and scope of Fibroblast Growth Factor signaling is immense, with documentation of its role in almost every organism and system studied so far. FGF ligands signal through a family of four distinct tyrosine kinase receptors, the FGF receptors (FGFRs). One contribution to the diversity of function and signaling of FGFs and their receptors arises from the numerous alternative splicing variants that have been documented in the FGFR literature. The present review discusses the types and roles of alternatively spliced variants of the FGFR family members and the significant impact of alternative splicing on the physiological functions of five broad classes of FGFR isoforms. Some characterized known regulatory mechanisms of alternative splicing and future directions in studies of FGFR alternative splicing are also discussed. Presence, absence, and/or the combination of specific exons within each FGFR protein impart upon each individual isoform its unique function and expression pattern during normal function and in diseased states (e.g., in cancers and birth defects). A better understanding of the diversity of FGF signaling in different developmental contexts and diseased states can be achieved through increased knowledge of the presence of specific FGFR isoforms and their impact on downstream signaling and functions. Modern high-throughput techniques afford an opportunity to explore the distribution and function of isoforms of FGFR during development and in diseases.

  7. p53 isoforms change p53 paradigm

    OpenAIRE

    2014-01-01

    Although p53 defines cellular responses to cancer treatment it is not clear how p53 can be used to control cell fate outcome. Data demonstrate that so-called p53 does not exist as a single protein, but is in fact a group of p53 protein isoforms whose expression can be manipulated to control the cellular response to treatment.

  8. Absolute quantitation of protein posttranslational modification isoform.

    Science.gov (United States)

    Yang, Zhu; Li, Ning

    2015-01-01

    Mass spectrometry has been widely applied in characterization and quantification of proteins from complex biological samples. Because the numbers of absolute amounts of proteins are needed in construction of mathematical models for molecular systems of various biological phenotypes and phenomena, a number of quantitative proteomic methods have been adopted to measure absolute quantities of proteins using mass spectrometry. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with internal peptide standards, i.e., the stable isotope-coded peptide dilution series, which was originated from the field of analytical chemistry, becomes a widely applied method in absolute quantitative proteomics research. This approach provides more and more absolute protein quantitation results of high confidence. As quantitative study of posttranslational modification (PTM) that modulates the biological activity of proteins is crucial for biological science and each isoform may contribute a unique biological function, degradation, and/or subcellular location, the absolute quantitation of protein PTM isoforms has become more relevant to its biological significance. In order to obtain the absolute cellular amount of a PTM isoform of a protein accurately, impacts of protein fractionation, protein enrichment, and proteolytic digestion yield should be taken into consideration and those effects before differentially stable isotope-coded PTM peptide standards are spiked into sample peptides have to be corrected. Assisted with stable isotope-labeled peptide standards, the absolute quantitation of isoforms of posttranslationally modified protein (AQUIP) method takes all these factors into account and determines the absolute amount of a protein PTM isoform from the absolute amount of the protein of interest and the PTM occupancy at the site of the protein. The absolute amount of the protein of interest is inferred by quantifying both the absolute amounts of a few PTM

  9. Aquaporin family genes exhibit developmentally-regulated and host-dependent transcription patterns in the sea louse Caligus rogercresseyi.

    Science.gov (United States)

    Farlora, Rodolfo; Valenzuela-Muñoz, Valentina; Chávez-Mardones, Jacqueline; Gallardo-Escárate, Cristian

    2016-07-01

    Aquaporins are small integral membrane proteins that function as pore channels for the transport of water and other small solutes across the cell membrane. Considering the important roles of these proteins in several biological processes, including host-parasite interactions, there has been increased research on aquaporin proteins recently. The present study expands on the knowledge of aquaporin family genes in parasitic copepods, examining diversity and expression during the ontogeny of the sea louse Caligus rogercresseyi. Furthermore, aquaporin expression was evaluated during the early infestation of Atlantic (Salmo salar) and Coho salmon (Oncorhynchus kisutch). Deep transcriptome sequencing data revealed eight full length and two partial open reading frames belonging to the aquaporin protein family. Clustering analyses with identified Caligidae sequences revealed three major clades of aquaglyceroporins (Cr-Glp), classical aquaporin channels (Cr-Bib and Cr-PripL), and unorthodox aquaporins (Cr-Aqp12-like). In silico analysis revealed differential expression of aquaporin genes between developmental stages and between sexes. Male-biased expression of Cr-Glp1_v1 and female-biased expression of Cr-Bib were further confirmed in adults by RT-qPCR. Additionally, gene expressions were measured for seven aquaporins during the early infestation stage. The majority of aquaporin genes showed significant differential transcription expressions between sea lice parasitizing different hosts, with Atlantic salmon sea lice exhibiting overall reduced expression as compared to Coho salmon. The observed differences in the regulation of aquaporin genes may reveal osmoregulatory adaptations associated with nutrient ingestion and metabolite waste export, exposing complex host-parasite relationships in C. rogercresseyi.

  10. Differential CARM1 Isoform Expression in Subcellular Compartments and among Malignant and Benign Breast Tumors.

    Directory of Open Access Journals (Sweden)

    David Shlensky

    Full Text Available Coactivator-associated arginine methyltransferase 1 (CARM1 is a coactivator for ERα and cancer-relevant transcription factors, and can methylate diverse cellular targets including histones. CARM1 is expressed in one of two alternative splice isoforms, full-length CARM1 (CARM1FL and truncated CARM1 (CARM1ΔE15. CARM1FL and CARM1ΔE15 function differently in transcriptional regulation, protein methylation, and mediation of pre-mRNA splicing in cellular models.To investigate the functional roles and the prognosis potential of CARM1 alternative spliced isoforms in breast cancer, we used recently developed antibodies to detect differential CARM1 isoform expression in subcellular compartments and among malignant and benign breast tumors.Immunofluorescence in MDA-MB-231 and BG-1 cell lines demonstrated that CARM1ΔE15 is the dominant isoform expressed in the cytoplasm, and CARM1FL is more nuclear localized. CARM1ΔE15 was found to be more sensitive to Hsp90 inhibition than CARM1FL, indicating that the truncated isoform may be the oncogenic form. Clinical cancer samples did not have significantly higher expression of CARM1FL or CARM1ΔE15 than benign breast samples at the level of mRNA or histology. Furthermore neither CARM1FL nor CARM1ΔE15 expression correlated with breast cancer molecular subtypes, tumor size, or lymph node involvement.The analysis presented here lends new insights into the possible oncogenic role of CARM1ΔE15. This study also demonstrates no obvious association of CARM1 isoform expression and clinical correlates in breast cancer. Recent studies, however, have shown that CARM1 expression correlates with poor prognosis, indicating a need for further studies of both CARM1 isoforms in a large cohort of breast cancer specimens.

  11. Antitumor effects in hepatocarcinoma of isoform-selective inhibition of HDAC2

    DEFF Research Database (Denmark)

    Lee, Yun-Han; Seo, Daekwan; Choi, Kyung-Ju

    2014-01-01

    Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit H...

  12. γIsoform-Selective Changes in PKC Immunoreactivity after Trace Eyeblink Conditioning in the Rabbit Hippocampus

    NARCIS (Netherlands)

    Zee, E.A. van der; Kronforst-Collins, M.A.; Maizels, E.T.; Hunzicker-Dunn, M.; Disterhoft, J.F.

    1997-01-01

    An immunocytochemical examination of the rabbit hippocampus was done to determine which of the Ca2+-dependent protein kinase C (PKC) isoforms (PKCα, -βI, -βII, or -γ) are involved in associative learning. The hippocampally dependent trace eyeblink conditioning task was used for behavioral training,

  13. PKC isoforms interact with and phosphorylate DNMT1

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    Pradhan Sriharsa

    2011-05-01

    Full Text Available Abstract Background DNA methyltransferase 1 (DNMT1 has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization. Results Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity. Conclusions Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.

  14. Crystallization and Identification of the Glycosylated Moieties of Two Isoforms of the Main Allergen Hev b 2 and Preliminary X-ray Analysis of Two Polymorphs of Isoform ll

    Energy Technology Data Exchange (ETDEWEB)

    Fuentes-Silva,D.; Mendoza-Hernandez, G.; Stojanoff, V.; Palomares, L.; Zenteno, E.; Torres-Larios, A.; Rodriguez-Romero, A.

    2007-01-01

    Latex from Hevea brasiliensis contains several allergenic proteins that are involved in type I allergy. One of them is Hev b 2, which is a {beta}-1,3-glucanase enzyme that exists in different isoforms with variable glycosylation content. Two glucanase isoforms were isolated from trees of the GV-42 clone by gel filtration, affinity and ion-exchange chromatography. Isoform I had a carbohydrate content of about 20%, with N-linked N-acetyl-glucosamine, N-acetyl-galactosamine, fucose and galactose residues as the main sugars, while isoform II showed 6% carbohydrate content consisting of N-acetyl-glucosamine, fucose, mannose and xylose. Both isoforms were crystallized by the hanging-drop vapor-diffusion method. Isoform I crystals were grown using 0.2 M trisodium citrate dihydrate, 0.1 M Na HEPES pH 7.5 and 20%(v/v) 2-propanol, but these crystals were not appropriate for data collection. Isoform II crystals were obtained under two conditions and X-ray diffraction data were collected from both. In the first condition (0.2 M trisodium citrate, 0.1 M sodium cacodylate pH 6.5, 30% 2-propanol), crystals belonging to the tetragonal space group P4{sub 1} with unit-cell parameters a = b = 150.17, c = 77.41 {angstrom} were obtained. In the second condition [0.2 M ammonium acetate, 0.1 M trisodium citrate dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] the isoform II crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 85.08, b = 89.67, c = 101.80 {angstrom}, {beta}= 113.6{sup o}. Preliminary analysis suggests that there are four molecules of isoform II in both asymmetric units.

  15. Water transport and functional dynamics of aquaporins in osmoregulatory organs of fishes.

    Science.gov (United States)

    Madsen, Steffen S; Engelund, Morten B; Cutler, Christopher P

    2015-08-01

    Aquaporins play distinct roles for water transport in fishes as they do in mammals-both at the cellular, organ, and organismal levels. However, with over 32,000 known species of fishes inhabiting almost every aquatic environment, from tidal pools, small mountain streams, to the oceans and extreme salty desert lakes, the challenge to obtain consensus as well as specific knowledge about aquaporin physiology in these vertebrate clades is overwhelming. Because the integumental surfaces of these animals are in intimate contact with the surrounding milieu, passive water loss and uptake represent two of the major osmoregulatory challenges that need compensation. However, neither obligatory nor regulatory water transport nor their mechanisms have been elucidated to the same degree as, for example, ion transport in fishes. Currently fewer than 60 papers address fish aquaporins. Most of these papers identify "what is present" and describe tissue expression patterns in various teleosts. The agnathans, chondrichthyans, and functionality of fish aquaporins generally have received little attention. This review emphasizes the functional physiology of aquaporins in fishes, focusing on transepithelial water transport in osmoregulatory organs in euryhaline species - primarily teleosts, but covering other taxonomic groups as well. Most current knowledge comes from teleosts, and there is a strong need for related information on older fish clades. Our survey aims to stimulate new, original research in this area and to bring together new collaborations across disciplines.

  16. Sparse linear modeling of next-generation mRNA sequencing (RNA-Seq) data for isoform discovery and abundance estimation.

    Science.gov (United States)

    Li, Jingyi Jessica; Jiang, Ci-Ren; Brown, James B; Huang, Haiyan; Bickel, Peter J

    2011-12-13

    Since the inception of next-generation mRNA sequencing (RNA-Seq) technology, various attempts have been made to utilize RNA-Seq data in assembling full-length mRNA isoforms de novo and estimating abundance of isoforms. However, for genes with more than a few exons, the problem tends to be challenging and often involves identifiability issues in statistical modeling. We have developed a statistical method called "sparse linear modeling of RNA-Seq data for isoform discovery and abundance estimation" (SLIDE) that takes exon boundaries and RNA-Seq data as input to discern the set of mRNA isoforms that are most likely to present in an RNA-Seq sample. SLIDE is based on a linear model with a design matrix that models the sampling probability of RNA-Seq reads from different mRNA isoforms. To tackle the model unidentifiability issue, SLIDE uses a modified Lasso procedure for parameter estimation. Compared with deterministic isoform assembly algorithms (e.g., Cufflinks), SLIDE considers the stochastic aspects of RNA-Seq reads in exons from different isoforms and thus has increased power in detecting more novel isoforms. Another advantage of SLIDE is its flexibility of incorporating other transcriptomic data such as RACE, CAGE, and EST into its model to further increase isoform discovery accuracy. SLIDE can also work downstream of other RNA-Seq assembly algorithms to integrate newly discovered genes and exons. Besides isoform discovery, SLIDE sequentially uses the same linear model to estimate the abundance of discovered isoforms. Simulation and real data studies show that SLIDE performs as well as or better than major competitors in both isoform discovery and abundance estimation. The SLIDE software package is available at https://sites.google.com/site/jingyijli/SLIDE.zip.

  17. Entropy-based model for miRNA isoform analysis.

    Directory of Open Access Journals (Sweden)

    Shengqin Wang

    Full Text Available MiRNAs have been widely studied due to their important post-transcriptional regulatory roles in gene expression. Many reports have demonstrated the evidence of miRNA isoform products (isomiRs in high-throughput small RNA sequencing data. However, the biological function involved in these molecules is still not well investigated. Here, we developed a Shannon entropy-based model to estimate isomiR expression profiles of high-throughput small RNA sequencing data extracted from miRBase webserver. By using the Kolmogorov-Smirnov statistical test (KS test, we demonstrated that the 5p and 3p miRNAs present more variants than the single arm miRNAs. We also found that the isomiR variant, except the 3' isomiR variant, is strongly correlated with Minimum Free Energy (MFE of pre-miRNA, suggesting the intrinsic feature of pre-miRNA should be one of the important factors for the miRNA regulation. The functional enrichment analysis showed that the miRNAs with high variation, particularly the 5' end variation, are enriched in a set of critical functions, supporting these molecules should not be randomly produced. Our results provide a probabilistic framework for miRNA isoforms analysis, and give functional insights into pre-miRNA processing.

  18. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Science.gov (United States)

    Chevreux, Sylviane; Solari, Pier Lorenzo; Roudeau, Stéphane; Deves, Guillaume; Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis; Ortega, Richard

    2009-11-01

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  19. EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Chevreux, Sylviane; Roudeau, Stephane; Deves, Guillaume; Ortega, Richard [Laboratoire de Chimie Nucleaire Analytique et Bioenvironnementale, CNRS UMR5084, Universite Bordeaux 1, Chemin du Solarium, F-33175 Gradignan cedex (France); Solari, Pier Lorenzo [Synchrotron SOLEIL, L' Orme des Merisiers, BP 48, F-91192 Gif-sur-Yvette cedex, Saint-Aubin (France); Alliot, Isabelle; Testemale, Denis; Hazemann, Jean Louis, E-mail: ortega@cenbg.in2p3.f [FAME, ESRF, 6 rue Jules Horowitz, BP220, F-38043 Grenoble cedex (France)

    2009-11-15

    Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.

  20. Aquaporin-1 and aquaporin-3 expressions in the temporomandibular joint condylar cartilage after an experimentally induced osteoarthritis

    Institute of Scientific and Technical Information of China (English)

    MENG Juan-hong; MA Xu-chen; LI Zhi-min; WU Deng-cheng

    2007-01-01

    Background Over 70% of the total tissue weight in the cartilage matrix consists of water,and the early-stage osteoarthritic cartilage is characterized by swelling.Water transport in the cartilage matrix and across the membranes of chondrocytes may be important in normal and pathological conditions of cartilage.The purpose of this study was to identify aquaporin-1 (AQP1) and aquaporin-3 (AQP3) expressions in the mandibular condylar cartilage after experimentally induced osteoarthritis(OA)in rats.Methods An experimental temporomandibular joint OA was induced by partial discectomy in rats.The pathological characteristics of the normal,early-stage,and late-stage osteoarthritic TMJ cartilages were verified by histological techniques.The AQP1 and AQP3 gene expressions in the normal and osteoarthritic cartilages were measured using quantitative real-time reverse-transcription PCR analysis.The cartilage sections were incubated in primary polyclonal antibodies to AQP3;immunofluorescent microscopy was used to examine the AQP3 expression shown by its protein level.Results The mRNA expression levels of AQP1 and AQP3,analyzed using quantitative PCR,revealed that AQP3 mRNA was highly up-regulated in the OA cartilage,which was considered significant.There was no notable difference in the expression of AQP1 mRNA between OA and normal controls.With the progressing of the OA,the localization of the AQP3 protein was quite different from that of the normal cartilage.Cormpared to the normal cartilage,the expressions of AQP3 protein were observed mainly in the proliferative zone and the upper mid-zone chondrocytes at the early-stage of OA,and were observed to appear frequently throughout the mid-and deep zone during the late-stage of OA.Conclusions The high expression of AQP3 mRNA in the OA cartilage and the different localization of the AQP3 protein suggest that it may play a particular role in OA pathogenesis.Further study of AQP3 function may provide new insight into the

  1. Aquaporin water channels in the canine gubernaculum testis.

    Science.gov (United States)

    Arrighi, Silvana; Aralla, Marina; Fracassetti, Paola; Mobasheri, Ali; Cremonesi, Fausto

    2013-07-01

    The jelly-like gubernaculum testis (GT) is a hydrated structure consisting of a concentric sheath of dense connective tissue around a loose mesenchymal core, with two cords of skeletal muscle cells asymmetrically placed alongside. Expansion of the GT occurs during the transabdominal phase of testicular descent, linked to cell proliferation together with modifications of the hydric content of the organ. The aim of this study was to detect immunohistochemically the presence of aquaporins (AQPs), integral membrane proteins permitting passive transcellular water movement, in the canine GTs. Samples (n=15) were obtained from pregnancies of 9 medium sized bitches and dissected from healthy fetuses. Five fetuses were aged 35-45 days of gestation, 10 fetuses from 46 days of gestation to delivery, thus offering us the opportunity to study the progressive maturation of the gubernacula. The presence of AQP3, 4, 7, 8 and -9 was assessed in the muscular components of the GT, some of them (AQP3, AQP4, AQP7) with increasing intensity through the second half of pregnancy up to term. AQP1 was localized in the capillary and venous endothelia in the younger fetuses, also in the artery adventitia and in the nerve perineurium in progressively older fetuses. These data demonstrate the potential importance and contribution of AQP-mediated water flux in hydration and volume modification of the growing GT in a canine model.

  2. Diabetes Insipidus in Mice with a Mutation in Aquaporin-2.

    Directory of Open Access Journals (Sweden)

    2005-08-01

    Full Text Available Congenital nephrogenic diabetes insipidus (NDI is a disease characterized by failure of the kidney to concentrate urine in response to vasopressin. Human kindreds with nephrogenic diabetes insipidus have been found to harbor mutations in the vasopressin receptor 2 (Avpr2 gene or the vasopressin-sensitive water channel aquaporin-2 (Aqp2 gene. Development of a treatment is rendered difficult due to the lack of a viable animal model. Through forward genetic screening of ethylnitrosourea-mutagenized mice, we report the identification and characterization of a mouse model of NDI, with an F204V mutation in the Aqp2 gene. Unlike previously attempted murine models of NDI, our mice survive to adulthood and more exactly recapitulate the human disorder. Previous in vitro experiments using renal cell lines suggest recessive Aqp2 mutations result in improper trafficking of the mutant water pore. Using these animals, we have directly proven this hypothesis of improper AQP2 translocation as the molecular defect in nephrogenic diabetes insipidus in the intact organism. Additionally, using a renal cell line we show that the mutated protein, AQP2-F204V, is retained in the endoplasmic reticulum and that this abnormal localization can be rescued by wild-type protein. This novel mouse model allows for further mechanistic studies as well as testing of pharmacological and gene therapies for NDI.

  3. Aquaporin 9 in rat brain after severe traumatic brain injury

    Directory of Open Access Journals (Sweden)

    Hui Liu

    2012-03-01

    Full Text Available OBJECTIVE: To reveal the expression and possible roles of aquaporin 9 (AQP9 in rat brain, after severe traumatic brain injury (TBI. METHODS: Brain water content (BWC, tetrazolium chloride staining, Evans blue staining, immunohistochemistry (IHC, immunofluorescence (IF, western blot, and real-time polymerase chain reaction were used. RESULTS: The BWC reached the first and second (highest peaks at 6 and 72 hours, and the blood brain barrier (BBB was severely destroyed at six hours after the TBI. The worst brain ischemia occurred at 72 hours after TBI. Widespread AQP9-positive astrocytes and neurons in the hypothalamus were detected by means of IHC and IF after TBI. The abundance of AQP9 and its mRNA increased after TBI and reached two peaks at 6 and 72 hours, respectively, after TBI. CONCLUSIONS: Increased AQP9 might contribute to clearance of excess water and lactate in the early stage of TBI. Widespread AQP9-positive astrocytes might help lactate move into neurons and result in cellular brain edema in the later stage of TBI. AQP9-positive neurons suggest that AQP9 plays a role in energy balance after TBI.

  4. Expression of aquaporin 8 and its relationship with melanosis coli

    Institute of Scientific and Technical Information of China (English)

    LIU Jun; TIAN De-an; WANG Jun-ping; ZHANG Su-zhen; FENG Jing; ZHAO Zhi-zhong; HAO Yu-xia; LIU Ping

    2011-01-01

    Background The relationship between melanosis coli (MC) and aquaporin 8 (AQP8) has not yet been elucidated. The aim of this research was to investigate the relationship between the expression of AQP8 and the pathological mechanism of MC.Methods Expression of AQP8 was detected by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) in 37 MC colon tissues and 13 control colon tissues. Global gene expression analysis was also used to identify differently expressed genes. Its relationship with MC was analyzed by SPSS 11.5 statistical software.Results The positive rate of AQP8 expression detected by immunohistochemistry in the MC group was 24.3% (9/37),significantly lower than the 69.2% (9/13) in the control group (P <0.05). The relative expression level of AQP8 in MC group was 0.639±0.160, lower than 0.921±0.148 of controls (P <0.05). Global gene expression analysis showed that AQP8 mRNA expression was downregulated in MC patients.Conclusions The decreased AQP8 expression in MC patients indicates that chronic use of laxatives containing anthraquinone may cause reduced water absorption. The expression of AQP8 may be related to MC.

  5. A molecular understanding of the dynamic mechanism of aquaporin osmosis

    CERN Document Server

    Shua, Liangsuo; Qian, Xin; Wanga, Xiyun; Lin, Yixin; Tan, Kai; Shu, Chaohui; Jin, Shiping

    2014-01-01

    AQPs (aquaporins), the rapid water channels of cells, play a key role in maintaining osmotic equilibrium of cells. In this paper, we reported the dynamic mechanism of AQP osmosis at the molecular level. A theoretical model based on molecular dynamics was carried out and verified by the published experimental data. The reflection coefficients ({\\sigma}) of neutral molecules are mainly decided by their relative size with AQPs, and increase with a third power up to a constant value 1. This model also indicated that the reflection coefficient of a complete impermeable solute can be smaller than 1. The H+ concentration of solution can influence the driving force of the AQPs by changing the equivalent diameters of vestibules surrounded by loops with abundant polar amino acids. In this way, pH of solution can regulate water permeability of AQPs. Therefore, an AQP may not only work as a switch to open or close, but as a rapid response molecular valve to control its water flow. The vestibules can prevent the channel b...

  6. Effects of shenmai injection on pulmonary aquaporin 1 in rats following traumatic brain injury

    Institute of Scientific and Technical Information of China (English)

    BAI Yu; YAO Hai-xia; HU Ming-lun; WANG Liang-rong; JIN Li-da; WANG Wan-tie; LIN Li-na

    2011-01-01

    Background Aquaporin-1 (AQP1) has involved in fluid transport in diverse pulmonary edema diseases. Our study aimed to explore the dynamic changes of AQP1 in pulmonary water metabolism in rats following traumatic brain injury (TBI) and the protective effect provided by shenmai injection.Methods Sixty male Sprague Dawley rats weighting 280-300 g were randomly divided into three groups: the normal control group, the model group and the shenrnai injection (SMI) group. One piece skull was taken away without injuring cerebral tissue in normal control group, while rats in model group and SMI group were subject to free fall injury in the cerebral hemisphere. Rats in model group received intraperitoneal normal sodium (15 mi/kg) at one hour post-injury and the same dose of shenmai injection instead in SMI group, respectively. The expression of AQP1 was detected by immunohistochemical analysis and semi-quantitative RT-PCR at 0 hour, 10 hours, 72 hours and 120 hours after TBI.Arterial blood gas analysis and lung wet to dry were also measured.Results AQP1 was mainly presented in the capillary endothelium and slightly alveolar epithelial cells in three groups, but the expression of AQP1 in the normal control group was positive and tenuous, weakly positive in the model and SMI groups,respectively. Compared with normal control group, AQP1 Mrna levels were down regulated in the model and SMI groups at 10 hours, 72 hours and 120 hours (P0.05).Conclusions The decreased AQP1 expression may be involved in the increased lung water content and dysfunction of pulmonary water metabolism following TBI. The treatment with SMI could improve water metabolism by promoting AQP1 expression.

  7. Aquaporin 8 expression is reduced and regulated by microRNAs in patients with ulcerative colitis

    Institute of Scientific and Technical Information of China (English)

    MIN Min; PENG Li-hua; SUN Gang; GUO Ming-zhou; QIU Ze-wu; YANG Yun-sheng

    2013-01-01

    Background Ulcerative colitis (UC) is associated with differential expression of genes involved in inflammation and tissue remodeling.MicroRNA (miRNA) plays an important role in the pathogenesis of UC by regulating the gene expression at the post-transcriptional level and control crucial physiological processes.This study aimed to identify aquaporin 8 (AQP8) expression and its relationship with miRNA in UC patients.Methods Human colon samples,in this study,were obtained from 20 patients with UC and 16 healthy subjects undergoing diagnostic colonoscopy at the Chinese People's Liberation Army General Hospital between December 2009and June 2010.We screened different genes from UC tissues and healthy subjects using genome-wide microarray,quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting.Regulation of gene expression by miRNAs was assessed by luciferase reporter construct assays and transfection of specific miRNA mimics and inhibitor.Results We identified that 1596 genes were increased and 1301 genes were decreased in UC patients compared to healthy subjects.Among them,we focused on the analysis of AQP8 which was decreased three folds in UC tissues (P <0.01).The expression of AQP8 mRNA and protein were decreased in UC tissue and tumor necrosis factor (TNF)-αtreated HT29 cells compared with controls (P <0.05).We searched candidate target miRNAs of AQP8 through bioformatics and the luciferase report assay analysis indicated that miR-424,miR-195,miR-330,miR-612,and miR-16 which has complementary site in the 3-untranslated region (3'UTR) of AQP8 could decrease the relative luciferase activities by 10%-45%.Conclusion AQP8 and its relationship with miRNAs may be involved in the pathogenesis of UC.

  8. Functional studies of sodium pump isoforms

    DEFF Research Database (Denmark)

    Clausen, Michael Jakob

    The Na+,K+-ATPase is an essential ion pump found in all animal cells. It uses the energy from ATP hydrolysis to export three Na+ and import two K+, both against their chemical gradients and for Na+ also against the electrical potential. Mammals require four Na+,K+-ATPase isoforms that each have u...... synthesized cohorts of pumps from the Golgi apparatus to the plasma membrane....

  9. Differential expression of aquaporin 3 in Triturus italicus from larval to adult epidermal conversion

    Directory of Open Access Journals (Sweden)

    E Brunelli

    2009-06-01

    Full Text Available By using immunohistochemical techniques applied to confocal microscopy, the presence of aquaporin 3 water channel in the epidermis of Triturus italicus (Amphibia, Urodela has been shown. We analysed the expression of aquaporin 3 (AQP3 during the larval, pre-metamorphic and adult phases; we also showed the localization of the water-channel protein AQP3 in free-swimming conditions and during aestivation in parallel with histological analysis of the skin, focusing on the possible relationship between protein expression and terrestrial habitats. Our results indicate that aquaporin is produced as the epidermis modifies during the functional maturation phase starting at the climax. Moreover, our data suggest an increase in enzyme expression in aestivating newts emphasizing the putative functional importance of differential expression related to a distinct phase of the biological cycle.

  10. The epsilon isoform of 14-3-3 protein is a component of the prion protein amyloid deposits of Gerstmann-Sträussler-Scheinker disease.

    Science.gov (United States)

    Di Fede, Giuseppe; Giaccone, Giorgio; Limido, Lucia; Mangieri, Michela; Suardi, Silvia; Puoti, Gianfranco; Morbin, Michela; Mazzoleni, Giulia; Ghetti, Bernardino; Tagliavini, Fabrizio

    2007-02-01

    The 14-3-3 proteins are highly conserved, ubiquitous molecules involved in a variety of biologic events, such as transduction pathway modulation, cell cycle control, and apoptosis. Seven isoforms have been identified that are abundant in the brain, preferentially localized in neurons. Remarkable increases in 14-3-3 are seen in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease (CJD), and it has been found in pathologic inclusions of several neurodegenerative diseases. Moreover, the zeta isoform has been detected in prion protein (PrP) amyloid deposits of CJD patients. To further investigate the cerebral distribution of 14-3-3 in prion-related encephalopathies, we carried out an immunohistochemical and biochemical analysis of brain tissue from patients with Gerstmann-Sträussler-Scheinker disease (GSS) and sporadic, familial and acquired forms of CJD, using specific antibodies against the seven 14-3-3 isoforms. The study showed a strong immunoreactivity of PrP amyloid plaques of GSS patients for the 14-3-3 epsilon isoform, but not for the other isoforms. The epsilon isoform of 14-3-3 was not found in PrP deposits of CJD. These results indicate that the epsilon isoform of 14-3-3 is a component of PrP amyloid deposits of GSS and suggest that this is the sole 14-3-3 isoform specifically involved in the neuropathologic changes associated with this disorder.

  11. FSH isoform pattern in classic galactosemia.

    Science.gov (United States)

    Gubbels, Cynthia S; Thomas, Chris M G; Wodzig, Will K W H; Olthaar, André J; Jaeken, Jaak; Sweep, Fred C G J; Rubio-Gozalbo, M Estela

    2011-04-01

    Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns of five classic galactosemia patients with POI were compared to the pattern obtained in two patients with a primary glycosylation disorder (phosphomannomutase-2-deficient congenital disorders of glycosylation, PMM2-CDG) and POI, and in five postmenopausal women as controls. We used FPLC chromatofocussing with measurement of FSH concentration per fraction, and discovered that there were no significant differences between galactosemia patients, PMM2-CDG patients and postmenopausal controls. Our results do not support that FSH dysfunction due to a less acidic isoform pattern because of hypoglycosylation is a key mechanism of POI in this disease.

  12. Aquaporin 1 and aquaporin 4 overexpression in bovine spongiform encephalopathy in a transgenic murine model and in cattle field cases.

    Science.gov (United States)

    Costa, Carme; Tortosa, Raül; Rodríguez, Agustín; Ferrer, Isidre; Torres, Juan Maria; Bassols, Anna; Pumarola, Martí

    2007-10-17

    Aquaporins (AQP) are a family of transmembrane proteins that act as water selective channels. AQP1 and AQP4 are widely expressed in the central nervous system where they play several roles. Overexpression of AQP has been reported in some human and animal transmissible spongiform encephalopathies, but information is scanty about their distribution in the central nervous system in bovine spongiform encephalopathy (BSE). Double immunohistochemistry for AQP1, AQP4 and GFAP was developed in a transgenic mouse line overexpressing the bovine cellular prion protein (BoTg110), intracerebrally infected with cattle BSE. Western blot for AQP1 and AQP4, and immunohistochemistry for both AQP and GFAP were carried out in cases of BSE-diagnosed cattle as part of surveillance plan in Catalonia (Spain). A marked increase in AQP1 and AQP4 was observed in mice at the terminal stage of the disease, when they had a wide range of clinical signs, whereas no increase could be observed in the early stage before the onset of the clinical signs. In cattle which did not show evidence of clinical signs, both AQP already showed a great increase. The AQP overexpression correlated with GFAP-immunoreactive astrocytes and PrPres deposition in both cases. The results of this study suggest that AQP overexpression in glial cells could lead to an imbalance in water and ion homeostasis which could contribute to triggering the typical histopathological changes of BSE.

  13. High-fat diet induced isoform changes of the Parkinson's disease protein DJ-1.

    Science.gov (United States)

    Poschmann, Gereon; Seyfarth, Katrin; Besong Agbo, Daniela; Klafki, Hans-Wolfgang; Rozman, Jan; Wurst, Wolfgang; Wiltfang, Jens; Meyer, Helmut E; Klingenspor, Martin; Stühler, Kai

    2014-05-02

    Genetic and environmental factors mediate via different physiological and molecular processes a shifted energy balance leading to overweight and obesity. To get insights into the underlying processes involved in energy intake and weight gain, we compared hypothalamic tissue of mice kept on a high-fat or control diet for 10 days by a proteomic approach. Using two-dimensional difference gel electrophoresis in combination with LC-MS/MS, we observed significant abundance changes in 15 protein spots. One isoform of the protein DJ-1 was elevated in the high-fat diet group in three different mouse strains SWR/J, C57BL/6N, and AKR/J analyzed. Large-scale validation of DJ-1 isoforms in individual samples and tissues confirmed a shift in the pattern of DJ-1 isoforms toward more acidic isoforms in several brain and peripheral tissues after feeding a high-fat diet for 10 days. The identification of oxidation of cysteine 106 as well as 2-succinyl modification of the same residue by mass spectrometry not only explains the isoelectric shift of DJ-1 but also links our results to similar shifts of DJ-1 observed in neurodegenerative disease states under oxidative stress. We hypothesize that DJ-1 is a common physiological sensor involved in both nutrition-induced effects and neurodegenerative disease states.

  14. Functional involvement of Annexin-2 in cAMP induced AQP2 trafficking.

    NARCIS (Netherlands)

    Tamma, G.; Procino, G.; Mola, M.G.; Svelto, M.; Valenti, G.

    2008-01-01

    Annexin-2 is required for the apical transport in epithelial cells. In this study, we investigated the involvement of annexin-2 in cAMP-induced aquaporin-2 (AQP2) translocation to the apical membrane in renal cells. We found that the cAMP-elevating agent forskolin increased annexin-2 abundance in th

  15. Isoform-specific anti-MeCP2 antibodies confirm that expression of the e1 isoform strongly predominates in the brain [v1; ref status: indexed, http://f1000r.es/1mg

    Directory of Open Access Journals (Sweden)

    Lara Kaddoum

    2013-10-01

    Full Text Available Rett syndrome is a neurological disorder caused by mutations in the MECP2 gene.  MeCP2 transcripts are alternatively spliced to generate two protein isoforms (MeCP2_e1 and MeCP2_e2 that differ at their N-termini. Whilst mRNAs for both forms are expressed ubiquitously, the one for MeCP2_e1 is more abundant than for MeCP2_e2 in the central nervous system. In transfected cells, both protein isoforms are nuclear and colocalize with densely methylated heterochromatic foci. With a view to understanding the physiological contribution of each isoform, and their respective roles in the pathogenesis of Rett syndrome, we set out to generate isoform-specific anti-MeCP2 antibodies. To this end, we immunized rabbits against the peptides corresponding to the short amino-terminal portions that are different between the two isoforms. The polyclonal antibodies thus obtained specifically detected their respective isoforms of MeCP2 in Neuro2a (N2A cells transfected to express either form. Both antisera showed comparable sensitivities when used for Western blot or immunofluorescence, and were highly specific for their respective isoform. When those antibodies were used on mouse tissues, specific signals were easily detected for Mecp2_e1, whilst Mecp2_e2 was very difficult to detect by Western blot, and even more so by immunofluorescence. Our results thus suggest that brain cells express low amounts of the Mecp2-e2 isoform. Our findings are compatible with recent reports showing that MeCP2_e2 is dispensable for healthy brain function, and that it may be involved in the regulation of neuronal apoptosis and embryonic development.

  16. Membrane domain specificity in the spatial distribution of aquaporins 5, 7, 9, and 11 in efferent ducts and epididymis of rats.

    Science.gov (United States)

    Hermo, Louis; Schellenberg, Morgan; Liu, Lauren Ye; Dayanandan, Bama; Zhang, Tong; Mandato, Craig A; Smith, Charles E

    2008-12-01

    Water content within the epididymis of the male reproductive system is stringently regulated to promote sperm maturation. Several members of the aquaporin (AQP) family of water channel-forming integral membrane proteins have been identified in epididymal cells, but expression profiling for this epithelium is presently incomplete, and no AQP isoform has yet been identified on basolateral plasma membranes of these cells. In this study, we explored AQP expression by RT-PCR and light microscopy immunolocalizations using peroxidase and wide-field fluorescence techniques. The results indicate that several AQPs are coexpressed in the epididymis including AQP 5, 7, 9, and 11. Immunolocalizations suggested complex patterns in the spatial distribution of these AQPs. In principal cells, AQP 9 and 11 were present mainly on microvilli, whereas AQP 7 was localized primarily to lateral and then to basal plasma membranes in a region-specific manner. AQP 5 was also expressed regionally but was associated with membranes of endosomes. Additionally, AQPs were expressed by some but not all basal (AQP 7 and 11), clear (AQP 7 and 9), and halo (AQP 7 and 11) cells. These findings indicate unique associations of AQPs with specific membrane domains in a cell type- and region-specific manner within the epididymis of adult animals.

  17. Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration

    Institute of Scientific and Technical Information of China (English)

    Qi Chen; Hongying Peng; Li Lei; Ying Zhang; Haibin Kuang; Yujing Cao; Qi-xian Shi; Tonghui Ma; Enkui Duan

    2011-01-01

    In the journey from the male to female reproductive tract,mammalian sperm experience a natural osmotic decrease (e.g.,in mouse,from ~415 mOsm in the cauda epididymis to ~310 mOsm in the uterine cavity). Sperm have evolved to utilize this hypotonic exposure for motility activation,meanwhile efficiently silence the negative impact of hypotonic cell swelling. Previous physiological and pharmacological studies have shown that ion channel-controlled water influx/efflux is actively involved in the process of sperm volume regulation; however,no specific sperm proteins have been found responsible for this rapid osmoadaptation. Here,we report that aquaporin3 (AQP3) is a sperm water channel in mice and humans. Aqp3-deficient sperm show normal motility activation in response to hypotonicity but display increased vulnerability to hypotonic cell swelling,characterized by increased tail bending after entering uterus. The sperm defect is a result of impaired sperm volume regulation and progressive cell swelling in response to physiological hypotonic stress during male-female reproductive tract transition. Time-lapse imaging revealed that the cell volume expansion begins at cytoplasmic droplet,forcing the tail to angulate and form a hairpin-like structure due to mechanical membrane stretch. The tail deformation hampered sperm migration into oviduct,resulting in impaired fertilization and reduced male fertility. These data suggest AQP3 as an essential membrane pathway for sperm regulatory volume decrease (RVD) that balances the "trade-off" between sperm motility and cell swelling upon physiological hypotonicity,thereby optimizing postcopulatory sperm behavior.

  18. The water channel aquaporin-1 contributes to renin cell recruitment during chronic stimulation of renin production.

    Science.gov (United States)

    Tinning, Anne R; Jensen, Boye L; Schweda, Frank; Machura, Katharina; Hansen, Pernille B L; Stubbe, Jane; Gramsbergen, Jan Bert; Madsen, Kirsten

    2014-12-01

    Both the processing and release of secretory granules involve water movement across granule membranes. It was hypothesized that the water channel aquaporin (AQP)1 directly contributes to the recruitment of renin-positive cells in the afferent arteriole. AQP1(-/-) and AQP1(+/+) mice were fed a low-salt (LS) diet [0.004% (wt/wt) NaCl] for 7 days and given enalapril [angiotensin-converting enzyme inhibitor (ACEI), 0.1 mg/ml] in drinking water for 3 days. There were no differences in plasma renin concentration at baseline. After LS-ACEI, plasma renin concentrations increased markedly in both genotypes but was significantly lower in AQP1(-/-) mice compared with AQP1(+/+) mice. Tissue renin concentrations were higher in AQP1(-/-) mice, and renin mRNA levels were not different between genotypes. Mean arterial blood pressure was not different at baseline and during LS diet but decreased significantly in both genotypes after the addition of ACEI; the response was faster in AQP1(-/-) mice but then stabilized at a similar level. Renin release after 200 μl blood withdrawal was not different. Isoprenaline-stimulated renin release from isolated perfused kidneys did not differ between genotypes. Cortical tissue norepinephrine concentrations were lower after LS-ACEI compared with baseline with no difference between genotypes. Plasma nitrite/nitrate concentrations were unaffected by genotype and LS-ACEI. In AQP1(-/-) mice, the number of afferent arterioles with recruitment was significantly lower compared with AQP1(+/+) mice after LS-ACEI. We conclude that AQP1 is not necessary for acutely stimulated renin secretion in vivo and from isolated perfused kidneys, whereas recruitment of renin-positive cells in response to chronic stimulation is attenuated or delayed in AQP1(-/-) mice.

  19. Tolerance checkpoint bypass permits emergence of pathogenic T cells to neuromyelitis optica autoantigen aquaporin-4

    Science.gov (United States)

    Sagan, Sharon A.; Winger, Ryan C.; Cruz-Herranz, Andrés; Nelson, Patricia A.; Hagberg, Sarah; Miller, Corey N.; Spencer, Collin M.; Ho, Peggy P.; Bennett, Jeffrey L.; Levy, Michael; Levin, Marc H.; Verkman, Alan S.; Steinman, Lawrence; Green, Ari J.; Anderson, Mark S.; Sobel, Raymond A.; Zamvil, Scott S.

    2016-01-01

    Aquaporin-4 (AQP4)-specific T cells are expanded in neuromyelitis optica (NMO) patients and exhibit Th17 polarization. However, their pathogenic role in CNS autoimmune inflammatory disease is unclear. Although multiple AQP4 T-cell epitopes have been identified in WT C57BL/6 mice, we observed that neither immunization with those determinants nor transfer of donor T cells targeting them caused CNS autoimmune disease in recipient mice. In contrast, robust proliferation was observed following immunization of AQP4-deficient (AQP4−/−) mice with AQP4 peptide (p) 135–153 or p201–220, peptides predicted to contain I-Ab–restricted T-cell epitopes but not identified in WT mice. In comparison with WT mice, AQP4−/− mice used unique T-cell receptor repertoires for recognition of these two AQP4 epitopes. Donor T cells specific for either determinant from AQP4−/−, but not WT, mice induced paralysis in recipient WT and B-cell–deficient mice. AQP4-specific Th17-polarized cells induced more severe disease than Th1-polarized cells. Clinical signs were associated with opticospinal infiltrates of T cells and monocytes. Fluorescent-labeled donor T cells were detected in CNS lesions. Visual system involvement was evident by changes in optical coherence tomography. Fine mapping of AQP4 p201–220 and p135–153 epitopes identified peptides within p201–220 but not p135–153, which induced clinical disease in 40% of WT mice by direct immunization. Our results provide a foundation to evaluate how AQP4-specific T cells contribute to AQP4-targeted CNS autoimmunity (ATCA) and suggest that pathogenic AQP4-specific T-cell responses are normally restrained by central tolerance, which may be relevant to understanding development of AQP4-reactive T cells in NMO. PMID:27940915

  20. Molecular machinery for vasotocin-dependent transepithelial water movement in amphibians: aquaporins and evolution.

    Science.gov (United States)

    Suzuki, Masakazu; Shibata, Yuki; Ogushi, Yuji; Okada, Reiko

    2015-08-01

    Amphibians represent the first vertebrates to adapt to terrestrial environments, and are successfully distributed around the world. The ventral skin, kidney, and urinary bladder are important osmoregulatory organs for adult anuran amphibians. Water channel proteins, called aquaporins (AQPs), play key roles in transepithelial water absorption/reabsorption in these organs. At least 43 types of AQPs were identified in anurans; a recent phylogenetic analysis categorized anuran AQPs among 16 classes (AQP0-14, 16). Anuran-specific AQPa2 was assigned to AQP6, then was further subdivided into the ventral skin-type (AQP6vs; AQPa2S), whose expression is confined to the ventral skin, and the urinary bladder-type (AQP6ub; AQPa2U), which is basically expressed in the urinary bladder. For the osmoregulatory organs, AQP3 is constitutively located in the basolateral plasma membrane of tight-junctioned epithelial cells. AQP6vs, AQP2 and/or AQP6ub are also expressed in these epithelial cells and are translocated to the apical membrane in response to arginine vasotocin, thereby regulating water absorption/reabsorption. It was suggested recently that two subtypes of AQP6vs contribute to cutaneous water absorption in Ranid species. In addition, AQP5 (AQP5a) and AQP5L (AQP5b) were identified from Xenopus tropicalis Gray, 1864, and AQP5 was localized to the apical membrane of luminal epithelial cells of the urinary bladder in dehydrated Xenopus. This finding suggested that AQP5 may be involved in water reabsorption from this organ under dehydration. Based on the hitherto reported information, we propose models for the evolution of water-absorbing/reabsorbing mechanisms in anuran osmoregulatory organs in association with AQPs.

  1. Aliskiren Increases Aquaporin-2 Expression and Attenuates Lithium-induced Nephrogenic Diabetes Insipidus.

    Science.gov (United States)

    Lin, Yu; Zhang, Tiezheng; Feng, Pinning; Qiu, Miaojuan; Liu, Qiaojuan; Li, Suchun; Zheng, Peili; Kong, Yonglun; Levi, Moshe; Li, Chunling; Wang, Weidong

    2017-02-22

    The direct renin inhibitor aliskiren has been shown to retain and persist in medullary collecting ducts even after treatment was discontinued, suggesting a new mechanism of action for this drug. The purpose of the present study was to investigate whether aliskiren regulates renal aquaporin expression in the collecting ducts and improves urinary concentrating defect induced by lithium in mice. The mice were either fed with normal chow or LiCl diet (40mM/kg dry food/day for 4 days and 20mM/kg dry food/day for last 3 days) for seven days. Some mice were intraperitoneally injected with aliskiren (50mg/kg BW/day in saline). Aliskiren significantly increased protein abundance of AQP2 in the kidney inner medulla in mice. In inner medulla collecting duct cell suspension, aliskiren markedly increased AQP2 and pS256-AQP2 protein abundance which was significantly inhibited either by adenylyl cyclase inhibitor MDL-12330A or by PKA inhibitor H89, indicating an involvement of the cAMP-PKA signaling pathway in aliskiren-induced increased AQP2 expression. Aliskiren treatment improved urinary concentrating defect in lithium-treated mice, and partially prevented the decrease of AQP2 and pS256-AQP2 protein abundance in inner medulla of the kidney. In conclusion, the direct renin inhibitor aliskiren upregulates AQP2 protein expression in inner medullary collecting duct principal cells and prevents lithium-induced nephrogenic diabetes insipidus (NDI) likely via cAMP-PKA pathways.

  2. Intestinal fluid absorption in anadromous salmonids: importance of tight junctions and aquaporins

    Directory of Open Access Journals (Sweden)

    Kristina eSundell

    2012-09-01

    Full Text Available The anadromous salmonid life cycle includes both fresh water (FW and seawater (SW stages. The parr-smolt transformation (smoltification pre–adapt the fish to SW while still in FW. The osmoregulatory organs change their mode of action from a role of preventing water inflow in FW, to absorb ions to replace water lost by osmosis in SW. During smoltification, the drinking rate increases, in the intestine the ion and fluid transport increases and is further elevated after SW entry. In SW, the intestine absorbs ions to create an inwardly directed water flow which is accomplished by increased Na+,K+-ATPase (NKA activity in the basolateral membrane, driving ion absorption via ion channels and/or co-transporters. This review will aim at discussing the expression patterns of the ion transporting proteins involved in intestinal fluid absorption in the FW stage, during smoltification and after SW entry. Of equal importance for intestinal fluid absorption as the active absorption of ions, is the permeability of the epithelium to ions and water. During the smoltification the increase in NKA activity and water uptake in SW is accompanied by decreased paracellular permeability suggesting a redirection of the fluid movement from a paracellular route in FW, to a transcellular route in SW. Increased transcellular fluid absorption could be achieved by incorporation of aquaporins (AQPs into the enterocyte membranes and/or by a change in fatty acid profile of the enterocyte lipid bilayer. An increased incorporation of unsaturated fatty acids into the membrane phospholipids will increase water permeability by enhancing the fluidity of the membrane. A second aim of the present review is therefore to discuss the presence and regulation of expression of AQPs in the enterocyte membrane as well as to discuss the profile of fatty acids present in the membrane phospholipids during different stages of the salmonid lifecycle.

  3. Expression of the Astrocyte Water Channel Aquaporin-4 in the Mouse Brain

    Directory of Open Access Journals (Sweden)

    Jacqueline A. Hubbard

    2015-10-01

    Full Text Available Aquaporin-4 (AQP4 is a bidirectional water channel that is found on astrocytes throughout the central nervous system. Expression is particularly high around areas in contact with cerebrospinal fluid, suggesting that AQP4 plays a role in fluid exchange between the cerebrospinal fluid compartments and the brain. Despite its significant role in the brain, the overall spatial and region-specific distribution of AQP4 has yet to be fully characterized. In this study, we used Western blotting and immunohistochemical techniques to characterize AQP4 expression and localization throughout the mouse brain. We observed AQP4 expression throughout the forebrain, subcortical areas, and brainstem. AQP4 protein levels were highest in the cerebellum with lower expression in the cortex and hippocampus. We found that AQP4 immunoreactivity was profuse on glial cells bordering ventricles, blood vessels, and subarachnoid space. Throughout the brain, AQP4 was expressed on astrocytic end-feet surrounding blood vessels but was also heterogeneously expressed in brain tissue parenchyma and neuropil, often with striking laminar specificity. In the cerebellum, we showed that AQP4 colocalized with the proteoglycan brevican, which is synthesized by and expressed on cerebellar astrocytes. Despite the high abundance of AQP4 in the cerebellum, its functional significance has yet to be investigated. Given the known role of AQP4 in synaptic plasticity in the hippocampus, the widespread and region-specific expression pattern of AQP4 suggests involvement not only in fluid balance and ion homeostasis but also local synaptic plasticity and function in distinct brain circuits.

  4. Expression of the Astrocyte Water Channel Aquaporin-4 in the Mouse Brain

    Science.gov (United States)

    Hubbard, Jacqueline A.; Hsu, Mike S.; Seldin, Marcus M.

    2015-01-01

    Aquaporin-4 (AQP4) is a bidirectional water channel that is found on astrocytes throughout the central nervous system. Expression is particularly high around areas in contact with cerebrospinal fluid, suggesting that AQP4 plays a role in fluid exchange between the cerebrospinal fluid compartments and the brain. Despite its significant role in the brain, the overall spatial and region-specific distribution of AQP4 has yet to be fully characterized. In this study, we used Western blotting and immunohistochemical techniques to characterize AQP4 expression and localization throughout the mouse brain. We observed AQP4 expression throughout the forebrain, subcortical areas, and brainstem. AQP4 protein levels were highest in the cerebellum with lower expression in the cortex and hippocampus. We found that AQP4 immunoreactivity was profuse on glial cells bordering ventricles, blood vessels, and subarachnoid space. Throughout the brain, AQP4 was expressed on astrocytic end-feet surrounding blood vessels but was also heterogeneously expressed in brain tissue parenchyma and neuropil, often with striking laminar specificity. In the cerebellum, we showed that AQP4 colocalized with the proteoglycan brevican, which is synthesized by and expressed on cerebellar astrocytes. Despite the high abundance of AQP4 in the cerebellum, its functional significance has yet to be investigated. Given the known role of AQP4 in synaptic plasticity in the hippocampus, the widespread and region-specific expression pattern of AQP4 suggests involvement not only in fluid balance and ion homeostasis but also local synaptic plasticity and function in distinct brain circuits. PMID:26489685

  5. The 'selfish brain' is regulated by aquaporins and autophagy under nutrient deprivation.

    Science.gov (United States)

    Ye, Qiao; Wu, Yonghong; Gao, Yan; Li, Zhihui; Li, Weiguang; Zhang, Chenggang

    2016-05-01

    The brain maintains its mass and physiological functional capacity compared with other organs under harsh conditions such as starvation, a mechanism termed the 'selfish brain' theory. To further investigate this phenomenon, mice were examined following water and/or food deprivation. Although the body weights of the mice, the weight of the organs except the brain and blood glucose levels were significantly reduced in the absence of water and/or food, the brain weight maintained its original state. Furthermore, no significant differences in the water content of the brain or its energy balance were observed when the mice were subjected to water and/or food deprivation. To further investigate the mechanism underlying the brain maintenance of water and substance homeostasis, the expression levels of aquaporins (AQPs) and autophagy‑specific protein long‑chain protein 3 (LC3) were examined. During the process of water and food deprivation, no significant differences in the transcriptional levels of AQPs were observed. However, autophagy activity levels were initially stimulated, then suppressed in a time‑dependent manner. LC3 and AQPs have important roles for the survival of the brain under conditions of food and water deprivation, which provided further understanding of the mechanism underlying the 'selfish brain' phenomenon. Although not involved in the energy regulation of the 'selfish brain', AQPs were observed to have important roles in water and food deprivation, specifically with regards to the control of water content. Additionally, the brain exhibits an 'unselfish strategy' using autophagy during water and/or food deprivation. The present study furthered current understanding of the 'selfish brain' theory, and identified additional regulating target genes of AQPs and autophagy, with the aim of providing a basis for the prevention of nutrient shortage in humans and animals.

  6. Hyperosmolality-mediated peritoneal microvascular vasodilation is linked to aquaporin function.

    Science.gov (United States)

    Zakaria, El Rasheid; Althani, Asma; Fawzi, Ashraf A; Fituri, Omar M

    2014-01-01

    Glucose-based peritoneal dialysis (PD) solutions dilate the parietal and visceral peritoneal microvasculature by endothelium-dependent mechanisms that primarily involve hyperosmolality. This PD-mediated dilation occurs by active intracellular glucose uptake and adenosine Al receptor activation, and by hyperosmolality-stimulated glibenclamide-sensitive potassium channels. Both pathways invoke NO as a second messenger for vasodilation. We hypothesized that during crystalloid-induced osmosis, the osmotic water flux through the transendothelial water-exclusive aquaporin 1 (AQP1) channels is the primary mechanism whereby the endothelium is being stimulated to instigate hyperosmolality-driven vasodilation. Four microvascular levels (diameters in the range 6 - 100 microm) were visualized by intravital videomicroscopy of the terminal ileum in anesthetized rats. Microvascular diameters and flow were measured after topical exposure to a 5% hypertonic mannitol or 2.5% glucose-based PD solution, at baseline and after brief tissue pre-treatment (with 0.1% glutaraldehyde for 10 seconds) or after combined tissue pre-treatment and pharmacologic blockade of AQP1 with HgCl2 (100 micromol/L). Vascular endothelial integrity was verified by the response to acetylcholine (10(-4) mol/L) and sodium nitroprusside (10(-4) mol/L). The hyperosmolar solutions both caused rapid and sustained vasodilation at all microvascular levels, which was not altered by tissue pre-treatment. Inhibition of AQP1 completely abolished the mannitol-induced vasodilation and markedly attenuated the PD fluid-mediated vasodilation. Neither glutaraldehyde pre-treatment nor HgCl2 affected tissue integrity or endothelial cell function. We conclude that the peritoneal microvascular vasodilation caused by hyperosmolar PD fluid is instigated by the osmotic water flux through AQP1. Clinical PD solutions have components other than hyperosmolality that can induce endothelium-dependent peritoneal microvascular vasodilation

  7. Aquaporin-1 Facilitates Angiogenic Invasion in the Pathologic Neovasculature that Accompanies Cirrhosis

    Science.gov (United States)

    Huebert, Robert C.; Vasdev, Meher M.; Shergill, Uday; Das, Amitava; Huang, Bing Q.; Charlton MR, Michael R.; LaRusso, Nicholas F.; Shah, Vijay H.

    2010-01-01

    Increasing evidence suggests that hepatic fibrosis and pathologic angiogenesis are inter-dependent processes that occur in parallel. Endothelial cell invasion is requisite for angiogenesis and thus studies of the mechanisms governing liver endothelial cell (LEC) invasion during cirrhosis are of great importance. Emerging research implicates amoeboid-type motility and membrane blebbing as features that may facilitate invasion through matrix-rich microenvironments. Aquaporins (AQPs) are integral membrane water channels, recognized for their importance in epithelial secretion and absorption. However, recent studies also suggest links between water transport and cell motility / invasion. Therefore, the purpose of this study was to test the hypothesis that AQP-1 is involved in amoeboid motility and angiogenic invasion during cirrhosis. AQP-1 expression and localization was examined in normal and cirrhotic liver tissues derived from human and mouse. AQP-1 levels were modulated in LEC using retroviral overexpression or siRNA knockdown and functional effects on invasion, membrane blebbing dynamics, and osmotic water permeability were assayed. Results demonstrate that AQP-1 is up-regulated in the small, angiogenic, neo-vasculature within the fibrotic septa of cirrhotic liver. AQP-1 overexpression promotes FGF-induced dynamic membrane blebbing in LEC which is sufficient to augment invasion through extracellular matrix. Additionally, AQP-1 localizes to plasma membrane blebs where it increases osmotic water permeability and locally facilitates the rapid, trans-membrane flux of water. CONCLUSION AQP-1 enhances osmotic water permeability and FGF-induced dynamic membrane blebbing in LEC and thereby drives invasion and pathologic angiogenesis during cirrhosis PMID:20578142

  8. Aquaporin deletion in mice reduces intraocular pressure and aqueous fluid production.

    Science.gov (United States)

    Zhang, Duo; Vetrivel, L; Verkman, A S

    2002-06-01

    Aquaporin (AQP) water channels are expressed in the eye at sites of aqueous fluid production and outflow: AQP1 and AQP4 in nonpigmented ciliary epithelium, and AQP1 in trabecular meshwork endothelium. Novel methods were developed to compare aqueous fluid dynamics in wild-type mice versus mice lacking AQP1 and/or AQP4. Aqueous fluid production was measured by in vivo confocal microscopy after transcorneal iontophoretic introduction of fluorescein. Intraocular pressure (IOP), outflow, and anterior chamber compliance were determined from pressure measurements in response to fluid infusions using micropipettes. Aqueous fluid volume and [Cl(-)] were assayed in samples withdrawn by micropipettes. In wild-type mice (CD1 genetic background, age 4-6 wk), IOP was 16.0 +/- 0.4 mmHg (SE), aqueous fluid volume 7.2 +/- 0.3 microl, fluid production 3.6 +/- 0.2 microl/h, fluid outflow 0.36 +/- 0.06 microl/h/mmHg, and compliance 0.036 +/- 0.006 microl/mmHg. IOP was significantly decreased by up to 1.8 mmHg (P fluid production by up to 0.9 microl/h in age/litter-matched mice lacking AQP1 and/or AQP4 (outbred CD1 and inbred C57/bl6 genetic backgrounds). However, AQP deletion did not significantly affect outflow, [Cl(-)], volume, or compliance. These results provide evidence for the involvement of AQPs in intraocular pressure regulation by facilitating aqueous fluid secretion across the ciliary epithelium. AQP inhibition may thus provide a novel approach for the treatment of elevated IOP.

  9. Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design.

    Science.gov (United States)

    Gilbreth, Ryan N; Truong, Khue; Madu, Ikenna; Koide, Akiko; Wojcik, John B; Li, Nan-Sheng; Piccirilli, Joseph A; Chen, Yuan; Koide, Shohei

    2011-05-10

    Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a "monobody," a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with K(d) values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

  10. Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design

    Energy Technology Data Exchange (ETDEWEB)

    Gilbreth, Ryan N.; Truong, Khue; Madu, Ikenna; Koide, Akiko; Wojcik, John B.; Li, Nan-Sheng; Piccirilli, Joseph A.; Chen, Yuan; Koide, Shohei (UC); (CHNMC)

    2011-07-25

    Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a 'monobody,' a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with K{sub d} values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

  11. Loss of desmoplakin isoform I causes early onset cardiomyopathy and heart failure in a Naxos‐like syndrome

    Science.gov (United States)

    Uzumcu, A; Norgett, E E; Dindar, A; Uyguner, O; Nisli, K; Kayserili, H; Sahin, S E; Dupont, E; Severs, N J; Leigh, I M; Yuksel‐Apak, M; Kelsell, D P; Wollnik, B

    2006-01-01

    Background Desmosomes are cellular junctions important for intercellular adhesion and anchoring the intermediate filament (IF) cytoskeleton to the cell membrane. Desmoplakin (DSP) is the most abundant desmosomal protein with 2 isoforms produced by alternative splicing. Methods We describe a patient with a recessively inherited arrhythmogenic dilated cardiomyopathy with left and right ventricular involvement, epidermolytic palmoplantar keratoderma, and woolly hair. The patient showed a severe heart phenotype with an early onset and rapid progression to heart failure at 4 years of age. Results A homozygous nonsense mutation, R1267X, was found in exon 23 of the desmoplakin gene, which results in an isoform specific truncation of the larger DSPI isoform. The loss of most of the DSPI specific rod domain and C‐terminal area was confirmed by Western blotting and immunofluorescence. We further showed that the truncated DSPI transcript is unstable, leading to a loss of DSPI. DSPI is reported to be an obligate constituent of desmosomes and the only isoform present in cardiac tissue. To address this, we reviewed the expression of DSP isoforms in the heart. Our data suggest that DSPI is the major cardiac isoform but we also show that specific compartments of the heart have detectable DSPII expression. Conclusions This is the first description of a phenotype caused by a mutation affecting only one DSP isoform. Our findings emphasise the importance of desmoplakin and desmosomes in epidermal and cardiac function and additionally highlight the possibility that the different isoforms of desmoplakin may have distinct functional properties within the desmosome. PMID:16467215

  12. Water transport and functional dynamics of aquaporins in osmoregulatory organs of fishes

    DEFF Research Database (Denmark)

    Madsen, Steffen S; Engelund, Morten B; Cutler, Christopher P

    2015-01-01

    salty desert lakes, the challenge to obtain consensus as well as specific knowledge about aquaporin physiology in these vertebrate clades is overwhelming. Because the integumental surfaces of these animals are in intimate contact with the surrounding milieu, passive water loss and uptake represent two...

  13. LIP5 interacts with aquaporin 2 and facilitates its lysosomal degradation.

    NARCIS (Netherlands)

    Balkom, B.W.M. van; Boone, M.; Hendriks, G.; Kamsteeg, E.J.; Robben, J.H.; Stronks, H.C.; Voorde, A. van der; Herp, F. van; Sluijs, P. van der; Deen, P.M.T.

    2009-01-01

    Vasopressin binding to the V2 receptor in renal principal cells leads to activation of protein kinase A, phosphorylation of aquaporin 2 (AQP2) at Ser256, and the translocation of AQP2 to the apical membrane, resulting in concentration of the urine. In contrast, phorbol ester-induced activation of pr

  14. Phosphorylation of rat aquaporin-4 at Ser(111) is not required for channel gating

    DEFF Research Database (Denmark)

    Assentoft, Mette; Kaptan, Shreyas; Fenton, Robert A

    2013-01-01

    Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 has been described as an important entry and exit site for water during formation of brain edema and regulation of AQP4 is theref...

  15. The aquaporin-2 water channel in autosomal dominant primary nocturnal enuresis.

    NARCIS (Netherlands)

    Deen, P.M.T.; Dahl, N.; Caplan, M.J.

    2002-01-01

    PURPOSE: Nocturnal enuresis is one of the most common diagnoses in a pediatric clinic. Recently, linkage analysis revealed a 2-point lod score of 4.2 in 6 families with dominant primary nocturnal enuresis around the aquaporin-2 (AQP2) water channel locus. Since primary nocturnal enuresis is ameliora

  16. Water fluxes through aquaporin-9 prime epithelial cells for rapid wound healing

    DEFF Research Database (Denmark)

    Karlsson, T.; Lagerholm, B. C.; Vikstrom, E.;

    2013-01-01

    Cells move along surfaces both as single cells and multi-cellular units. Recent research points toward pivotal roles for water flux through aquaporins (AQPs) in single cell migration. Their expression is known to facilitate this process by promoting rapid shape changes. However, little is known...

  17. Enhancement of proton conductance by mutations of the selectivity filter of aquaporin-1

    DEFF Research Database (Denmark)

    Li, Hui; Chen, Hanning; Steinbronn, Christina;

    2011-01-01

    Prevention of cation permeation in wild-type aquaporin-1 (AQP1) is believed to be associated with the Asn-Pro-Ala (NPA) region and the aromatic/arginine selectivity filter (SF) domain. Previous work has suggested that the NPA region helps to impede proton permeation due to the protein backbone co...

  18. Concerted action of two cation filters in the aquaporin water channel

    DEFF Research Database (Denmark)

    Wu, Binghua; Steinbronn, Christina; Alsterfjord, Magnus;

    2009-01-01

    Aquaporin (AQP) facilitated water transport is common to virtually all cell membranes and is marked by almost perfect specificity and high flux rates. Simultaneously, protons and cations are strictly excluded to maintain ionic transmembrane gradients. Yet, the AQP cation filters have not been ide...

  19. Aquaporin-1 Expression in Retinal Pigment Epithelial Cells Overlying Retinal Drusen

    DEFF Research Database (Denmark)

    Tran, Thuy Linh; Bek, Toke; la Cour, Morten

    2016-01-01

    PURPOSE: In the outer retina, age-related macular degeneration (AMD) results in reduced hydraulic conductivity in Bruch's membrane, possibly leading to altered water transport in retinal pigment epithelial (RPE) cells. We hypothesize that RPE cells may express aquaporin-1 (AQP1) to compensate...

  20. Immunogenic potential of Rhipicephalus (Boophilus) microplus aquaporin 1 against Rhipicephalus sanguineus in domestic dogs

    Science.gov (United States)

    This study evaluated a recombinant aquaporin 1 protein of Rhipicephalus microplus (RmAQP1) as antigen in a vaccine against R. sanguineus. Five dogs were vaccinated with RmAQP1 (10 µg) + adjuvant (Montanide) (G1), and five were inoculated with adjuvant only (G2), three times. Twenty-one days after th...

  1. Role of cytoplasmic termini in sorting and shuttling of the aquaporin-2 water channel

    NARCIS (Netherlands)

    Balkom, B.W.M. van; Graat, M.P.J.; Raak, M.M.J.P. van; Hofman, E.; Sluijs, P. van der; Deen, P.M.T.

    2004-01-01

    In mammals, the regulation of water homeostasis is mediated by the aquaporin-1 (AQP1) water channel, which localizes to the basolateral and apical membranes of the early nephron segment, and AQP2, which is translocated from intracellular vesicles to the apical membrane of collecting duct cells after

  2. Aquaporin 2 and apical calcium-sensing receptor: new players in polyuric disorders associated with hypercalciuria.

    NARCIS (Netherlands)

    Procino, G.; Mastrofrancesco, L.; Mira, A.; Tamma, G.; Carmosino, M.; Emma, F.; Svelto, M.; Valenti, G.

    2008-01-01

    The kidney plays a critical role in regulating water homeostasis through specific proteins highly expressed in the kidney, called aquaporins, allowing water permeation at a high rate. This brief review focuses on some nephropathies associated with impaired urinary concentrating ability and in partic

  3. Functional challenge affects aquaporin mRNA abundance in mouse blastocysts

    DEFF Research Database (Denmark)

    Offenberg, Hanne Kjær; Thomsen, Preben Dybdahl

    2005-01-01

    The aquaporins (AQPs) are a family of channel proteins that facilitate diffusion of water across cell membranes. Three members of the AQP family have been detected in the mouse blastocyst: AQP 3 and 8 are located in the basolateral domain and AQP 9 predominantly in the apical domain...

  4. Aquaporins are major determinants of water use efficiency of rice plants in the field.

    Science.gov (United States)

    Nada, Reham M; Abogadallah, Gaber M

    2014-10-01

    This study aimed at specifying the reasons of unbalanced water relations of rice in the field at midday which results in slowing down photosynthesis and reducing water use efficiency (WUE) in japonica and indica rice under well-watered and droughted conditions. Leaf relative water content (RWC) decreased in the well-watered plants at midday in the field, but more dramatically in the droughted indica (75.6 and 71.4%) than japonica cultivars (85.5 and 80.8%). Gas exchange was measured at three points during the day (9:00, 13:00 and 17:00). Leaf internal CO2 (Ci) was not depleted when midday stomatal depression was highest indicating that Ci was not limiting to photosynthesis. Most aquaporins were predominantly expressed in leaves suggesting higher water permeability in leaves than in roots. The expression of leaf aquaporins was further induced by drought at 9:00 without comparable responses in roots. The data suggest that aquaporin expression in the root endodermis was limiting to water uptake. Upon removal of the radial barriers to water flow in roots, transpiration increased instantly and photosynthesis increased after 4h resulting in increasing WUE after 4h, demonstrating that WUE in rice is largely limited by the inadequate aquaporin expression profiles in roots.

  5. Multicentre comparison of a diagnostic assay: Aquaporin-4 antibodies in neuromyelitis optica

    NARCIS (Netherlands)

    P. Waters (Patrick); M. Reindl (Markus); A. Saiz (Albert Abe); K. Schanda (Kathrin); F. Tuller (Friederike); V. Kral (Vlastimil); P. Nytrova (Petra); O. Sobek (Ondrej); H.H. Nielsen (Helle Hvilsted); T. Barington (Torben); S.T. Lillevang (Søren T.); Z. Illes (Zsolt); K. Rentzsch (Kristin); A. Berthele (Achim); T. Berki (Tímea); L. Granieri; A. Bertolotto (Antonio); B. Giometto; L. Zuliani (Luigi); D. Hamann (Dörte); J.L. Van Pelt (Joost L.); R.Q. Hintzen (Rogier); R. Höftberger (Romana); C. Costa (Carme); M. Comabella (Manuel); X. Montalban (Xavier); M. Tintoré; A. Siva (Aksel); A. Altintas (Ayse); G. Deniz (Gunnur); M. Woodhall (Mark); J. Palace (Jacqueline); F. Paul (Friedemann); H.P. Hartung; O. Aktas (Orhan); S. Jarius (Sven); B. Wildemann (Brigitte); C. Vedeler (Christian); A. Ruiz (Anne); M.I. Leite (M. Isabel); P. Trillenberg (Peter); M. Probst (Monika); S. Saschenbrecker (Sandra); A.J.P.E. Vincent (Arnoud); R. Marignier (Romain)

    2016-01-01

    textabstractObjective Antibodies to cell surface central nervous system proteins help to diagnose conditions which often respond to immunotherapies. The assessment of antibody assays needs to reflect their clinical utility. We report the results of a multicentre study of aquaporin (AQP) 4 antibody (

  6. Bidirectional water fluxes and specificity for small hydrophilic molecules in aquaporins 0-5

    DEFF Research Database (Denmark)

    Meinild, A K; Klærke, Dan Arne; Zeuthen, T

    1998-01-01

    The dimensions of the aqueous pore in aquaporins (AQP) 0, 1, 2, 3, 4, and 5 expressed in Xenopus laevisoocytes were probed by comparing the ability of various solutes to generate osmotic flow. By improved techniques, volume flows were determined from initial rates of changes. Identical values...

  7. Androgen receptor isoforms in human and rat prostate

    Institute of Scientific and Technical Information of China (English)

    Shu-JieXIA; Gang-YaoHAO; Xiao-DaTANG

    2000-01-01

    Aim: To investigate the androgen receptor (AR) isoforms and its variability of expression in human and rat prostatic tissues. Methods: Human benign prostatic hyperplasia (BPH) and prostatic cancer tissues were obtained from patients undergoing prostatectomy, and rat ventral prostate was incised 3 days after castration. Forty-one AR-positive BPH specimens, 3 prostatic cancer specimens, and 6 rat prostates were used. After processing at 4℃, the tissues were examined by means of high resolution isoelectric focusing (IEF) technique to determine their AR isoforms. Results:From the prostatic specimens, 3 types of AR isoforms were detected with pI values at 6.5, 6.0, and 5.3. In human BPH tissues, 15/41 (36.6%) specimens showed all the three types of isoforms, while 19/41 (46.3%) showed 2 isoforms at various combinations and 7/41(17.1%), 1 isoform. For the 3 prostatic cancer specimens, one showed 3 isoforms, one, 2 isoforms, and the other failed to show any isoform. All rat prostatic tissues showed 2 isoforms at different combinations. Binding of 3H-dihydrotestosterone (DHT) to the isoforms was inhibited by the addition of 100-fold excess of DHT or testosterone, but not progesterone, oestradiol or diethylstilboestrol. Conclusion: AR isoforms are different in different patients. Although their genesis is not clear, the therapeutic implication of the present observation appears to be interesting, that may help clarifying the individual differences in the response to hormonal therapy.(Asian J Androl 2000 Dec;2:307-310)

  8. The tonoplast intrinsic aquaporin (TIP) subfamily of Eucalyptus grandis: Characterization of EgTIP2, a root-specific and osmotic stress-responsive gene.

    Science.gov (United States)

    Rodrigues, Marcela I; Bravo, Juliana P; Sassaki, Flávio T; Severino, Fábio E; Maia, Ivan G

    2013-12-01

    Aquaporins have important roles in various physiological processes in plants, including growth, development and adaptation to stress. In this study, a gene encoding a root-specific tonoplast intrinsic aquaporin (TIP) from Eucalyptus grandis (named EgTIP2) was investigated. The root-specific expression of EgTIP2 was validated over a panel of five eucalyptus organ/tissues. In eucalyptus roots, EgTIP2 expression was significantly induced by osmotic stress imposed by PEG treatment. Histochemical analysis of transgenic tobacco lines (Nicotiana tabacum SR1) harboring an EgTIP2 promoter:GUS reporter cassette revealed major GUS staining in the vasculature and in root tips. Consistent with its osmotic-stress inducible expression in eucalyptus, EgTIP2 promoter activity was up-regulated by mannitol treatment, but was down-regulated by abscisic acid. Taken together, these results suggest that EgTIP2 might be involved in eucalyptus response to drought. Additional searches in the eucalyptus genome revealed the presence of four additional putative TIP coding genes, which could be individually assigned to the classical TIP1-5 groups.

  9. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation.

    Science.gov (United States)

    Pritchard, Rory A; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated.

  10. Gene interference regulates aquaporin-4 expression in swollen tissue of rats with cerebral ischemic edema Correlation with variation in apparent diffusion coefficient

    Institute of Scientific and Technical Information of China (English)

    Hui Hu; Hong Lu; Zhanping He; Xiangjun Han; Jing Chen; Rong Tu

    2012-01-01

    To investigate the effects of mRNA interference on aquaporin-4 expression in swollen tissue of rats with ischemic cerebral edema, and diagnose the significance of diffusion-weighted MRI, we injected 5 μL shRNA- aquaporin-4 (control group) or siRNA- aquaporin-4 solution (1:800) (RNA interference group) into the rat right basal ganglia immediately before occlusion of the middle cerebral artery. At 0.25 hours after occlusion of the middle cerebral artery, diffusion-weighted MRI displayed a high signal; within 2 hours, the relative apparent diffusion coefficient decreased markedly, aquaporin-4 expression increased rapidly, and intracellular edema was obviously aggravated; at 4 and 6 hours, the relative apparent diffusion coefficient slowly returned to control levels, aquaporin-4 expression slightly increased, and angioedema was observed. In the RNA interference group, during 0.25- 6 hours after injection of siRNA- aquaporin-4 solution, the relative apparent diffusion coefficient slightly fluctuated and aquaporin-4 expression was upregulated; during 0.5-4 hours, the relative apparent diffusion coefficient was significantly higher, while aquaporin-4 expression was significantly lower when compared with the control group, and intracellular edema was markedly reduced; at 0.25 and 6 hours, the relative apparent diffusion coefficient and aquaporin-4 expression were similar when compared with the control group; obvious angioedema remained at 6 hours. Pearson's correlation test results showed that aquaporin-4 expression was negatively correlated with the apparent diffusion coefficient (r = -0.806, P < 0.01). These findings suggest that upregulated aquaporin-4 expression is likely to be the main molecular mechanism of intracellular edema and may be the molecular basis for decreased relative apparent diffusion coefficient. Aquaporin-4 gene interference can effectively inhibit the upregulation of aquaporin-4 expression during the stage of intracellular edema with time

  11. A characterization of the expression of 14-3-3 isoforms in psoriasis, basal cell carcinoma, atopic dermatitis and contact dermatitis

    Directory of Open Access Journals (Sweden)

    Line Raaby

    2010-10-01

    Full Text Available 14-3-3 is a highly conserved protein involved in a number of cellular processes including cell signalling, cell cycle regulation and gene transcription. Seven isoforms of the protein have been identified; β, γ, ε, ζ, η, σ and τ. The expression profile of the various isoforms in skin diseases is unknown. To investigate the expression of the seven 14-3-3 isoforms in involved and uninvolved skin from psoriasis, basal cell carcinoma (BCC, atopic dermatitis and nickel induced allergic contact dermatitis. Punch biopsies from involved and uninvolved skin were analyzed with quantitative reverse transcription-polymerase chain reaction to determine the mRNA expression of the 14-3-3 isoforms. The protein level of 14-3-3 isoforms was measured by Western blot technique in keratome biopsies from patients with psoriasis. Evaluation of dermal and epidermal protein expression was performed by immunofluorescence staining. Increased 14-3-3τ mRNA levels were detected in involved skin from patients with psoriasis, contact dermatitis and BCC. 14-3-3σ mRNA expression was increased in psoriasis and contact dermatitis, but not in BCC. In atopic dermatitis no significant difference between involved and uninvolved skin was found. The expression of the 14-3-3 isoforms was also studied at the protein level in psoriasis. Only 14-3-3τ expression was significantly increased in involved psoriatic skin compared with uninvolved skin. Immuno­fluorescence staining with 14-3-3τ- and 14-3-3σ-specific antibodies showed localization of both isoforms to the cytoplasm of the keratinocytes in the various skin sections. These results demonstrate a disease specific expression profile of the 14-3-3τ and 14-3-3σ isoforms.

  12. FGFR3 isoforms have distinct functions in the regulation of growth and cell morphology.

    Science.gov (United States)

    Shimizu, Akio; Takashima, Yuji; Kurokawa-Seo, Misuzu

    2002-01-11

    We have previously cloned the alternatively spliced isoform of fibroblast growth factor receptor 3 (FGFR3DeltaAB) that lacks the acid box in the extracellular region. To understand the biological functions and signal transduction of these FGFR3 isoforms, we analyzed the effect of FGF1 in ATDC5 cells, chondroprogenitor cell lines overexpressing these isoforms. In response to FGF1, FGFR3 induced a marked cell-morphology change to a round shape, while FGFR3DeltaAB did not. Furthermore, FGFR3 induced complete growth arrest, whereas FGFR3DeltaAB induced only moderate growth inhibition. Both receptors induced the expression of the CDK inhibitor p21(CIP1). However, only FGFR3 induced STAT1 phosphorylation that mediates the transcriptional induction of p21(CIP1), although both FGFR3 isoforms could induce a strong activation of mitogen-activated protein (MAP) kinases. Taken together, the different biological responses mediated by FGFR3 and FGFR3DeltaAB appear to be due to a difference in their ability to utilize STAT1 pathway and signals involved in cell rounding.

  13. Identification of p53 and Its Isoforms in Human Breast Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Zorka Milićević

    2014-01-01

    Full Text Available In breast carcinoma, disruption of the p53 pathway is one of the most common genetic alterations. The observation that the p53 can express multiple protein isoforms adds a novel level of complexity to the outcome of p53 mutations. p53 expression was analysed by Western immunoblotting and immunohistochemistry using monoclonal antibodies DO-7, Pab240, and polyclonal antiserum CM-1. The more frequently p53-positive nuclear staining has been found in the invasive breast tumors. One of the most intriguing findings is that mutant p53 appears as discrete dot-shaped regions within the nucleus of breast cancer cells. In many malignant cells, the nucleolar sequestration of p53 is evident. These observations support the view that the nucleolus is involved directly in the mediation of p53 function or indirectly by the control of the localization of p53 interplayers. p53 expressed in the nuclear fraction of breast cancer cells revealed a wide spectrum of isoforms. p53 isoforms ΔNp53 (47 kDa and Δ133p53β (35 kDa, known as dominant-negative repressors of p53 function, were detected as the most predominant variants in nuclei of invasive breast carcinoma cells. The isoforms expressed also varied between individual tumors, indicating potential roles of these p53 variants in human breast cancer.

  14. Cloning and expression of progesterone receptor isoforms A and B in bovine corpus luteum.

    Science.gov (United States)

    Rekawiecki, Robert; Kowalik, Magdalena Karolina; Kotwica, Jan

    2015-09-01

    Progesterone (P4) affects a cell through its nuclear receptor (PGR), which has two main isoforms: A (PGRA) and B (PGRB). A partial section of previously unknown PGRB cDNA from cattle was cloned. Next, mRNA and protein levels for these two isoforms in corpora lutea (CL) collected during different stages of the oestrous cycle and pregnancy were determined. The PGRB mRNA level was highest on Days 2-5 of the oestrous cycle, decreased over the next few days (P0.05) and increased during Weeks 6-12 (P<0.05). PGRB protein concentration followed a similar pattern but at a markedly lower level. Both PGRA and PGRB isoforms are involved in the regulation of P4 action, especially in the newly formed CL and developed CL in the first trimester of pregnancy. These data suggest that the variable expression of these isoforms during the oestrous cycle may depend on the influence of P4.

  15. Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development.

    Directory of Open Access Journals (Sweden)

    Nathalie Schmieg

    Full Text Available Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.

  16. Insulin receptor isoform A ameliorates long-term glucose intolerance in diabetic mice.

    Science.gov (United States)

    Diaz-Castroverde, Sabela; Gómez-Hernández, Almudena; Fernández, Silvia; García-Gómez, Gema; Di Scala, Marianna; González-Aseguinolaza, Gloria; Fernández-Millán, Elisa; González-Rodríguez, Águeda; García-Bravo, María; Chambon, Pierre; Álvarez, Carmen; Perdomo, Liliana; Beneit, Nuria; Escribano, Oscar; Benito, Manuel

    2016-11-01

    Type 2 diabetes mellitus is a complex metabolic disease and its pathogenesis involves abnormalities in both peripheral insulin action and insulin secretion. Previous in vitro data showed that insulin receptor isoform A, but not B, favours basal glucose uptake through its specific association with endogenous GLUT1/2 in murine hepatocytes and beta cells. With this background, we hypothesized that hepatic expression of insulin receptor isoform A in a mouse model of type 2 diabetes could potentially increase the glucose uptake of these cells, decreasing the hyperglycaemia and therefore ameliorating the diabetic phenotype. To assure this hypothesis, we have developed recombinant adeno-associated viral vectors expressing insulin receptor isoform A (IRA) or isoform B (IRB) under the control of a hepatocyte--specific promoter. Our results demonstrate that in the long term, hepatic expression of IRA in diabetic mice is more efficient than IRB in ameliorating glucose intolerance. Consequently, it impairs the induction of compensatory mechanisms through beta cell hyperplasia and/or hypertrophy that finally lead to beta cell failure, reverting the diabetic phenotype in about 8 weeks. Our data suggest that long-term hepatic expression of IRA could be a promising therapeutic approach for the treatment of type 2 diabetes mellitus.

  17. Two putative-aquaporin genes are differentially expressed during arbuscular mycorrhizal symbiosis in Lotus japonicus

    Directory of Open Access Journals (Sweden)

    Giovannetti Marco

    2012-10-01

    Full Text Available Abstract Background Arbuscular mycorrhizas (AM are widespread symbioses that provide great advantages to the plant, improving its nutritional status and allowing the fungus to complete its life cycle. Nevertheless, molecular mechanisms that lead to the development of AM symbiosis are not yet fully deciphered. Here, we have focused on two putative aquaporin genes, LjNIP1 and LjXIP1, which resulted to be upregulated in a transcriptomic analysis performed on mycorrhizal roots of Lotus japonicus. Results A phylogenetic analysis has shown that the two putative aquaporins belong to different functional families: NIPs and XIPs. Transcriptomic experiments have shown the independence of their expression from their nutritional status but also a close correlation with mycorrhizal and rhizobial interaction. Further transcript quantification has revealed a good correlation between the expression of one of them, LjNIP1, and LjPT4, the phosphate transporter which is considered a marker gene for mycorrhizal functionality. By using laser microdissection, we have demonstrated that one of the two genes, LjNIP1, is expressed exclusively in arbuscule-containing cells. LjNIP1, in agreement with its putative role as an aquaporin, is capable of transferring water when expressed in yeast protoplasts. Confocal analysis have demonstrated that eGFP-LjNIP1, under its endogenous promoter, accumulates in the inner membrane system of arbusculated cells. Conclusions Overall, the results have shown different functionality and expression specificity of two mycorrhiza-inducible aquaporins in L. japonicus. One of them, LjNIP1 can be considered a novel molecular marker of mycorrhizal status at different developmental stages of the arbuscule. At the same time, LjXIP1 results to be the first XIP family aquaporin to be transcriptionally regulated during symbiosis.

  18. The lineage-specific evolution of aquaporin gene clusters facilitated tetrapod terrestrial adaptation.

    Directory of Open Access Journals (Sweden)

    Roderick Nigel Finn

    Full Text Available A major physiological barrier for aquatic organisms adapting to terrestrial life is dessication in the aerial environment. This barrier was nevertheless overcome by the Devonian ancestors of extant Tetrapoda, but the origin of specific molecular mechanisms that solved this water problem remains largely unknown. Here we show that an ancient aquaporin gene cluster evolved specifically in the sarcopterygian lineage, and subsequently diverged into paralogous forms of AQP2, -5, or -6 to mediate water conservation in extant Tetrapoda. To determine the origin of these apomorphic genomic traits, we combined aquaporin sequencing from jawless and jawed vertebrates with broad taxon assembly of >2,000 transcripts amongst 131 deuterostome genomes and developed a model based upon Bayesian inference that traces their convergent roots to stem subfamilies in basal Metazoa and Prokaryota. This approach uncovered an unexpected diversity of aquaporins in every lineage investigated, and revealed that the vertebrate superfamily consists of 17 classes of aquaporins (Aqp0 - Aqp16. The oldest orthologs associated with water conservation in modern Tetrapoda are traced to a cluster of three aqp2-like genes in Actinistia that likely arose >500 Ma through duplication of an aqp0-like gene present in a jawless ancestor. In sea lamprey, we show that aqp0 first arose in a protocluster comprised of a novel aqp14 paralog and a fused aqp01 gene. To corroborate these findings, we conducted phylogenetic analyses of five syntenic nuclear receptor subfamilies, which, together with observations of extensive genome rearrangements, support the coincident loss of ancestral aqp2-like orthologs in Actinopterygii. We thus conclude that the divergence of sarcopterygian-specific aquaporin gene clusters was permissive for the evolution of water conservation mechanisms that facilitated tetrapod terrestrial adaptation.

  19. Transcriptional profiles of glutathione-S-Transferase isoforms, Cyp, and AOE genes in atrazine-exposed zebrafish embryos.

    Science.gov (United States)

    Glisic, Branka; Hrubik, Jelena; Fa, Svetlana; Dopudj, Nela; Kovacevic, Radmila; Andric, Nebojsa

    2016-02-01

    Glutathione-S-transferase (GST) superfamily consists of multiple members involved in xenobiotic metabolism. Expressional pattern of the GST isoforms in adult fish has been used as a biomarker of exposure to environmental chemicals. However, GST transcriptional responses vary across organs, thus requiring a cross-tissue examination of multiple mRNAs for GST profiling in an animal after chemical exposure. Zebrafish embryos express all GST isoforms as adult fish and could therefore represent an alternative model for identification of biomarkers of exposure. To evaluate such a possibility, we studied a set of cytosolic and microsomal GST isoform-specific expression profiles in the zebrafish embryos after exposure to atrazine, a widely used herbicide. Expression of the GST isoforms was compared with that of CYP genes involved in the phase I of xenobiotic metabolism and antioxidant enzyme (AOE) genes. Using quantitative real-time PCR, we showed dynamic changes in the expressional pattern of twenty GST isoforms, cyp1a, cyp3a65, ahr2, and four AOEs in early development of zebrafish. Acute (48 and 72 h) exposure of 24 h-old embryos to atrazine, from environmentally relevant (0.005 mg/L) to high (40 mg/L) concentrations, caused a variety of transient, albeit minor changes (atrazine (5 and 40 mg/L). In summary, an analysis of the response of multiple systems in the zebrafish embryos provided a comprehensive understanding of atrazine toxicity and its potential impact on biological processes.

  20. PSD-95 mediates membrane clustering of the human plasma membrane Ca2+ pump isoform 4b

    OpenAIRE

    Padányi, Rita; Pászty, Katalin; STREHLER, EMANUEL E.; Enyedi, Ágnes

    2008-01-01

    Besides the control of global calcium changes, specific plasma membrane calcium ATPase (PMCA) isoforms are involved in the regulation of local calcium signals. Although local calcium signaling requires the confinement of signaling molecules into microdomains, little is known about the specific organization of PMCA molecules within the plasma membrane. Here we show that co-expression with the postsynaptic–density-95 (PSD-95) scaffolding protein increased the plasma membrane expression of PMCA4...

  1. Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform

    OpenAIRE

    María de los Milagros Cámara; Bouvier, León A.; Canepa, Gaspar E.; Mariana R Miranda; Pereira, Claudio A.

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas’ disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a fe...

  2. Isoforms of murine and human serum amyloid P component

    DEFF Research Database (Denmark)

    Nybo, Mads; Hackler, R; Kold, B;

    1998-01-01

    Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did not a...... treatment caused a shift of the isoforms, but no reduction in isoform number. Two-dimensional gel electrophoresis confirmed the existence of multiple isoforms of human SAP monomers.......Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did...... not affect their number. When the acute-phase response was analysed in three mouse strains, CBA/J and C3H/HeN initially showed seven SAP isoforms in serum and C57BL/6 J three or four. The responses in all three strains peaked at day 2 and were normalized within 14 days. On days 2 and 4, CBA/J and C3H...

  3. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light

    Science.gov (United States)

    Jagannath, Aarti; Hughes, Steven; Abdelgany, Amr; Pothecary, Carina A.; Di Pretoro, Simona; Pires, Susana S.; Vachtsevanos, Athanasios; Pilorz, Violetta; Brown, Laurence A.; Hossbach, Markus; MacLaren, Robert E.; Halford, Stephanie; Gatti, Silvia; Hankins, Mark W.; Wood, Matthew J.A.; Foster, Russell G.; Peirson, Stuart N.

    2015-01-01

    Summary Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light [1, 2] including circadian entrainment [3], sleep induction [4], the pupillary light response (PLR) [5], and negative masking of locomotor behavior (the acute suppression of activity in response to light) [6]. How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails [7]. Significantly, both isoforms form fully functional photopigments [7]. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors. PMID:26320947

  4. Contribution of nitric oxide synthase isoforms to cholinergic vasodilation in murine retinal arterioles.

    Science.gov (United States)

    Gericke, Adrian; Goloborodko, Evgeny; Sniatecki, Jan J; Steege, Andreas; Wojnowski, Leszek; Pfeiffer, Norbert

    2013-04-01

    Nitric oxide synthases (NOSs) are critically involved in regulation of ocular perfusion. However, the contribution of the individual NOS isoforms to vascular responses is unknown in the retina. Because some previous findings suggested an involvement of inducible nitric oxide synthase (iNOS) in the regulation of retinal vascular tone, a major goal of the present study was to examine the hypothesis that iNOS is involved in mediating cholinergic vasodilation responses of murine retinal arterioles. Another subject of this study was to test the contribution of the other two NOS isoforms, neuronal (nNOS) and endothelial NOS (eNOS), to cholinergic retinal arteriole responses. Expression of individual NOS isoforms was determined in murine retinal arterioles using real-time PCR. All three NOS isoforms were expressed in retinal arterioles. However, eNOS mRNA was found to be most, and iNOS mRNA least abundant. To examine the functional relevance of iNOS for mediating vascular responses, retinal vascular preparations from gene-targeted iNOS-deficient mice (iNOS-/-) and wild-type mice were studied in vitro. Changes in luminal vessel diameter in response to the thromboxane mimetic 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619), the endothelium-dependent vasodilator acetylcholine, and the nitric oxide donor nitroprusside were measured by video microscopy. To determine the contribution of individual NOS isoforms to cholinergic vasodilation responses, retinas from iNOS-/- and wild-type mice were incubated with Nω-nitro-l-arginine methyl ester (l-NAME), a non-isoform-selective inhibitor of NOS, 7-nitroindazole, a selective nNOS blocker and aminoguanidine, a selective iNOS inhibitor. U-46619 evoked concentration-dependent vasoconstriction that was similar in retinal arterioles from iNOS-/- and wild-type mice. In retinal arterioles preconstricted with U-46619, acetylcholine and nitroprusside produced dose-dependent dilation that did not differ between iNOS-/- and

  5. Serum anti-aquaporin 4 antibody in neuromyelitis patients is not correlated with the length of injured spinal cord segments

    Institute of Scientific and Technical Information of China (English)

    Shirong Li; Lan Chu; Shuai Dong; Hui Yu; Zhu Xu; Hao Wang

    2011-01-01

    Clinical information and serum samples of 20 neuromyelitis patients and 30 patients with multiple sclerosis were collected in this study. The expression of anti-aquaporin 4 antibody in the serum of all patients was detected with an indirect immunofluorescence assay, using human embryonic kidney 293 cell line that stably express human-derived aquaporin 4 as a substrate. The characteristics of head and spinal magnetic resonance imaging were also observed in patients who had neuromyelitis and were positive for anti-aquaporin 4 antibody. Results showed that the expression of anti-aquaporin 4 antibody was significantly different between multiple sclerosis patients and neuromyelitis patients. There were 13 out of 20 neuromyelitis patients (including high-risk syndrome) that were positive for anti-aquaporin 4 antibody. The magnetic resonance imaging examinations of the head and spinal cord found that among the 13 positive patients, nine cases showed normal cerebral hemisphere and optic nerve, two cases had optic nerve changes, and one case had an atypical lesion in the brain. All 30 multiple sclerosis patients were negative for this antibody. The experimental findings indicate that patients with neuromyelitis optica had more than three lesioned segments in the spinal cord by magnetic resonance imaging, and the segment length of the injured spinal cord was not associated with the titer of aquaporin 4 antibody in neuromyelitis patients.

  6. Localization and functional characterization of the human NKCC2 isoforms

    DEFF Research Database (Denmark)

    Carota, I; Theilig, F; Oppermann, M;

    2010-01-01

    AIM: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na(+)/K(+)/2Cl(-) cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2...... isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. METHODS: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were...... performed to characterize human NKCC2 isoforms. RESULTS: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant...

  7. Evidence for leptin receptor isoforms heteromerization at the cell surface.

    Science.gov (United States)

    Bacart, Johan; Leloire, Audrey; Levoye, Angélique; Froguel, Philippe; Jockers, Ralf; Couturier, Cyril

    2010-06-01

    Leptin mediates its metabolic effects through several leptin receptor (LEP-R) isoforms. In humans, long (LEPRb) and short (LEPRa,c,d) isoforms are generated by alternative splicing. Most of leptin's effects are believed to be mediated by the OB-Rb isoform. However, the role of short LEPR isoforms and the possible existence of heteromers between different isoforms are poorly understood. Using BRET1 and optimized co-immunoprecipitation, we observed LEPRa/b and LEPRb/c heteromers located at the plasma membrane and stabilized by leptin. Given the widespread coexpression of LEPRa and LEPRb, our results suggest that LEPRa/b heteromers may represent a major receptor species in most tissues.

  8. Modifying the NH2 and COOH termini of aquaporin-5: effects on localization in polarized epithelial cells.

    Science.gov (United States)

    Wellner, Robert B; Hong, Sohee; Cotrim, Ana P; Swaim, William D; Baum, Bruce J

    2005-01-01

    To reengineer polarized epithelial cell functions directly in situ, or ex vivo in the fabrication of an artificial organ, it is necessary to understand mechanisms that account for polarized membrane sorting. We have used the aquaporins (AQPs), a family of homotetrameric water channel proteins, as model membrane proteins for this purpose. AQP monomers contain six transmembrane-spanning domains linked by five interconnecting loops, with the NH2 and COOH termini residing in the cytosol. AQP5 is localized in the apical membranes of several different epithelia in vivo, and in stably transfected MDCK-II cells grown as a polarized monolayer. We wished to identify a structural region(s) within rat AQP5 (rAQP5) important for apical localization, and to study the MDCK-II cell localization of rAQP5s modified in either their NH2 or COOH terminus. We show that the NH2- terminal region does not play a major role in apical localization as deletion of the NH2 terminus produced a modified rAQP5 construct (AQP5-NT(del)) that was stably expressed and localized primarily to the apical membranes of MDCK-II cells. Attachment of a FLAG epitope to the NH2 terminus of AQP5 (AQP5(flag) construct) also did not perturb apical localization. In addition, we found that the exchange of NH2-terminal regions between rAQP5 and human AQP1 (hAQP1; a nonpolarized AQP isoform) produced a modified rAQP5 construct (AQP5-1NT) and a modified hAQP1 construct (AQP1-5NT), each of which localized as the parental AQP (apically, and to both apical and basolateral membranes, respectively). In contrast, we found that deletion of the COOH terminus resulted in a modified rAQP5 construct (AQP5-CT(del)) that was unstably expressed and localized to intracellular site(s) in MDCK-II cells. Substitution of the COOH terminus of AQP1 with the COOH terminus of AQP5 also produced a construct (AQP1-5CT) transiently expressed in intracellular compartment(s). However, substitution of the COOH terminus of rAQP5 with the COOH

  9. Reciprocity in the developmental regulation of aquaporins 1, 3 and 5 during pregnancy and lactation in the rat.

    Directory of Open Access Journals (Sweden)

    Sasan Nazemi

    Full Text Available Milk secretion involves significant flux of water, driven largely by synthesis of lactose within the Golgi apparatus. It has not been determined whether this flux is simply a passive consequence of the osmotic potential between cytosol and Golgi, or whether it involves regulated flow. Aquaporins (AQPs are membrane water channels that regulate water flux. AQP1, AQP3 and AQP5 have previously been detected in mammary tissue, but evidence of developmental regulation (altered expression according to the developmental and physiological state of the mammary gland is lacking and their cellular/subcellular location is not well understood. In this paper we present evidence of developmental regulation of all three of these AQPs. Further, there was evidence of reciprocity since expression of the rather abundant AQP3 and less abundant AQP1 increased significantly from pregnancy into lactation, whereas expression of the least abundant AQP5 decreased. It would be tempting to suggest that AQP3 and AQP1 are involved in the secretion of water into milk. Paradoxically, however, it was AQP5 that demonstrated most evidence of expression located at the apical (secretory membrane. The possibility is discussed that AQP5 is synthesized during pregnancy as a stable protein that functions to regulate water secretion during lactation. AQP3 was identified primarily at the basal and lateral membranes of the secretory cells, suggesting a possible involvement in regulated uptake of water and glycerol. AQP1 was identified primarily at the capillary and secretory cell cytoplasmic level and may again be more concerned with uptake and hence milk synthesis, rather than secretion. The fact that expression was developmentally regulated supports, but does not prove, a regulatory involvement of AQPs in water flux through the milk secretory cell.

  10. First Trimester Pregnancy Loss and the Expression of alternatively spliced NKp30 isoforms in Maternal Blood and Placental Tissue

    Directory of Open Access Journals (Sweden)

    Avishai eShemesh

    2015-06-01

    Full Text Available In this study, we aimed to investigate whether first trimester pregnancy loss is associated with differences in expression of NKp30 splice variants (isoforms in maternal peripheral blood or placental tissue. We conducted a prospective case-control study; a total of 33 women undergoing dilation and curettage due to first trimester pregnancy loss were further subdivided into groups with sporadic or recurrent pregnancy loss. The control group was comprised of women undergoing elective termination of pregnancy. The qPCR approach was employed to assess the relative expression of NKp30 isoforms as well as the total expression of NKp30 and NKp46 receptors between the selected groups. Results show that in both PBMC and placental tissue, NKp46 and NKp30 expression was mildly elevated in the pregnancy loss groups compared with the elective group. In particular, NKp46 elevation was significant. Moreover, expression analysis of NKp30 isoforms manifested a different profile between PBMC and the placenta. NKp30-a and NKp30-b isoforms in the placental tissue, but not in PBMC, showed a significant increase in the pregnancy loss groups compared with the elective group. Placental expression of NKp30 activating isoforms -a and -b in the pregnancy loss groups was negatively correlated with PLGF expression. In contrast, placental expression of these isoforms in the elective group was positively correlated with TNFα, IL-10 and VEGF-A expression. The altered expression of NKp30 activating isoforms in placental tissue from patients with pregnancy loss compared to the elective group and the different correlations with cytokine expression point to the involvement of NKp30-mediated function in pregnancy loss.

  11. Differential regulation of T cell antigen responsiveness by isoforms of the src-related tyrosine protein kinase p59fyn

    OpenAIRE

    1992-01-01

    Recent observations suggest that the src-related tyrosine protein kinase p59fyn may be involved in antigen-induced T lymphocyte activation. As a result of alternative splicing, p59fyn exists as two isoforms that differ exclusively within a short sequence spanning the end of the Src Homology 2 (SH2) region and the beginning of the tyrosine protein kinase domain. While one p59fyn isoform (fynB) is highly expressed in brain, the alternative product (fynT) is principally found in T lymphocytes. T...

  12. Engineering of insulin receptor isoform-selective insulin analogues.

    Directory of Open Access Journals (Sweden)

    Tine Glendorf

    Full Text Available BACKGROUND: The insulin receptor (IR exists in two isoforms, A and B, and the isoform expression pattern is tissue-specific. The C-terminus of the insulin B chain is important for receptor binding and has been shown to contact the IR just adjacent to the region where the A and B isoforms differ. The aim of this study was to investigate the importance of the C-terminus of the B chain in IR isoform binding in order to explore the possibility of engineering tissue-specific/liver-specific insulin analogues. METHODOLOGY/PRINCIPAL FINDINGS: Insulin analogue libraries were constructed by total amino acid scanning mutagenesis. The relative binding affinities for the A and B isoform of the IR were determined by competition assays using scintillation proximity assay technology. Structural information was obtained by X-ray crystallography. Introduction of B25A or B25N mutations resulted in analogues with a 2-fold preference for the B compared to the A isoform, whereas the opposite was observed with a B25Y substitution. An acidic amino acid residue at position B27 caused an additional 2-fold selective increase in affinity for the receptor B isoform for analogues bearing a B25N mutation. Furthermore, the combination of B25H with either B27D or B27E also resulted in B isoform-preferential analogues (2-fold preference even though the corresponding single mutation analogues displayed no differences in relative isoform binding affinity. CONCLUSIONS/SIGNIFICANCE: We have discovered a new class of IR isoform-selective insulin analogues with 2-4-fold differences in relative binding affinities for either the A or the B isoform of the IR compared to human insulin. Our results demonstrate that a mutation at position B25 alone or in combination with a mutation at position B27 in the insulin molecule confers IR isoform selectivity. Isoform-preferential analogues may provide new opportunities for developing insulin analogues with improved clinical benefits.

  13. Expression of aquaporin-1 in rat pleural mesothelial cells and its specific inhibition by RNA interference in vitro

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wei; XIE Can-mao; LI Zhi-ping

    2007-01-01

    Background The discovery of water channel aquaporins(AQPs)has greatly expanded the understanding of the regulation of the water permeability of biological membranes.Aquaporin-1(AQP1)may be involved in fluid transport in numerous pathological conditions.The objective of the present study was to examine whether AQP1 is present in cultured rat pleural mesothelial cells(PMCs)and to investigate the specific inhibitory effect of RNA interference(RNAi) on AQP1 expression in PMCs,which may provide a new method for the further studies on the relation between expression of AQP1 in PMCs and pleural fluid removal in vivo.Methods PMCs were isolated and cultured from rat pleura.The expression of AQP1 in PMCs was confirmed by immunocytochemical staining and reverse transcriptase-polymerase chain reaction(RT-PCR).Two eukaryotic expression plasmid vectors of short hairpin RNA(shRNA)specific for the AQP1 gene of rat sapien were designed and constructed.The recombinant plasmid vectors were transfected into cultured rat PMCs by cation liposomes.Flow cytometry was used to screen the most effective shRNA at 48 hours after transfection.The expressions of AQP1 mRNA and protein were detected by RT-PCR and Western blotting method at 48 hours after transfection.Results RT-PCR and immunostaining revealed that AQP1 mRNA and protein were present in cultured rat PMCs.Two effective eukaryotic expression plasmid vectors of shRNA specific for the AQP1 gene were constructed successfully.The levels of the expression of AQP1 were inhibited by 83.45%,90.93%,respectively,at mRNA level and 41.24%,67.60%,respectively at protein level by two recombinant plasmids at 48 hours after transfection.The expression of AQP1 in PMCs transfected with plasmid was significantly lower than that of the cells transfected with the control plasmid HK and that of the untransfected cells(P<0.01).There was no significant difference in AQP1 expression between the control group and the group transfected with AQP1 nonspecific sh

  14. Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis.

    Science.gov (United States)

    Pham, Phuong Anh; Wahl, Vanessa; Tohge, Takayuki; de Souza, Laise Rosado; Zhang, Youjun; Do, Phuc Thi; Olas, Justyna J; Stitt, Mark; Araújo, Wagner L; Fernie, Alisdair R

    2015-11-01

    The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds.

  15. Quantitative evaluation of alternatively spliced mRNA isoforms by label-free real-time plasmonic sensing.

    Science.gov (United States)

    Huertas, César S; Carrascosa, L G; Bonnal, S; Valcárcel, J; Lechuga, L M

    2016-04-15

    Alternative splicing of mRNA precursors enables cells to generate different protein outputs from the same gene depending on their developmental or homeostatic status. Its deregulation is strongly linked to disease onset and progression. Current methodologies for monitoring alternative splicing demand elaborate procedures and often present difficulties in discerning between closely related isoforms, e.g. due to cross-hybridization during their detection. Herein, we report a general methodology using a Surface Plasmon Resonance (SPR) biosensor for label-free monitoring of alternative splicing events in real-time, without any cDNA synthesis or PCR amplification requirements. We applied this methodology to RNA isolated from HeLa cells for the quantification of alternatively spliced isoforms of the Fas gene, involved in cancer progression through regulation of programmed cell death. We demonstrate that our methodology is isoform-specific, with virtually no cross-hybridization, achieving limits of detection (LODs) in the picoMolar (pM) range. Similar results were obtained for the detection of the BCL-X gene mRNA isoforms. The results were independently validated by RT-qPCR, with excellent concordance in the determination of isoform ratios. The simplicity and robustness of this biosensor technology can greatly facilitate the exploration of alternative splicing biomarkers in disease diagnosis and therapy.

  16. Two isoforms of TALDO1 generated by alternative translational initiation show differential nucleocytoplasmic distribution to regulate the global metabolic network

    Science.gov (United States)

    Moriyama, Tetsuji; Tanaka, Shu; Nakayama, Yasumune; Fukumoto, Masahiro; Tsujimura, Kenji; Yamada, Kohji; Bamba, Takeshi; Yoneda, Yoshihiro; Fukusaki, Eiichiro; Oka, Masahiro

    2016-01-01

    Transaldolase 1 (TALDO1) is a rate-limiting enzyme involved in the pentose phosphate pathway, which is traditionally thought to occur in the cytoplasm. In this study, we found that the gene TALDO1 has two translational initiation sites, generating two isoforms that differ by the presence of the first 10 N-terminal amino acids. Notably, the long and short isoforms were differentially localised to the cell nucleus and cytoplasm, respectively. Pull-down and in vitro transport assays showed that the long isoform, unlike the short one, binds to importin α and is actively transported into the nucleus in an importin α/β-dependent manner, demonstrating that the 10 N-terminal amino acids are essential for its nuclear localisation. Additionally, we found that these two isoforms can form homo- and/or hetero-dimers with different localisation dynamics. A metabolite analysis revealed that the subcellular localisation of TALDO1 is not crucial for its activity in the pentose phosphate pathway. However, the expression of these two isoforms differentially affected the levels of various metabolites, including components of the tricarboxylic acid cycle, nucleotides, and sugars. These results demonstrate that the nucleocytoplasmic distribution of TALDO1, modulated via alternative translational initiation and dimer formation, plays an important role in a wide range of metabolic networks. PMID:27703206

  17. One-step extraction of functional recombinant aquaporin Z from inclusion bodies with optimal detergent.

    Science.gov (United States)

    Wang, Lili; Zhou, Hu; Li, Zhengjun; Lim, Teck Kwang; Lim, Xin Shan; Lin, Qingsong

    2015-11-01

    Aquaporins are integral membrane channel proteins found in all kingdoms of life. The Escherichia coli aquaporin Z (AqpZ) has been shown to solely conduct water at high permeability. Functional AqpZ is generally purified from the membrane fraction. However, the quantity of the purified protein is limited. In this study, a new method is developed to achieve high yield of bioactive AqpZ protein. A mild detergent n-dodecyl-β-D-maltopyranoside (DDM) was used to solubilize the over-expressed insoluble AqpZ from inclusion bodies without a refolding process. The recovered AqpZ protein showed high water permeability comparable with AqpZ obtained from the membrane fraction. In this way, the total yield of bioactive AqpZ has been increased greatly, which will facilitate the structural and functional characterization and future applications of AqpZ.

  18. Use of Aquaporins to Achieve Needed Water Purity On ISS for the EMU Space Suit System

    Science.gov (United States)

    Hill, Terry R.; Taylor, Brandon W.

    2011-01-01

    With the U.S. Space Shuttle fleet retired, the supply of extremely high-quality water 'super-Q' - required for the EMU Space suit cooling on this ISS - will become a significant operational hardware challenge in the very near future. A proposed potential solution is the use of a filtration system consisting of a semi-permeable membrane embedded with aquaporin proteins. Aquaporins are a special class of trans-membrane proteins that facilitate passive transport of water and other substances across a membrane. The specificity of these proteins is such that only water is allowed through the protein structure, and this novel property invites their adaptation for use in water filtration systems, specifically usage on the ISS for the EMU space suit system. These proteins are found in many living systems and have been developed for commercial use today.

  19. Increased expression of aquaporin-4 in human traumatic brain injury and brain tumors

    Institute of Scientific and Technical Information of China (English)

    HU Hua; YAO Hong-tian; ZHANG Wei-ping; ZHANG LEI; DING Wei; ZHANG Shi-hong; CHEN Zhong; WEI Er-qing

    2005-01-01

    Objective: To characterize the expression of aquaporin-4 (AQP4), one of the aquaporins (AQPs), in human brain specimens from patients with traumatic brain injury or brain tumors. Methods: Nineteen human brain specimens were obtained from the patients with traumatic brain injury, brain tumors, benign meningioma or early stage hemorrhagic stroke. MRI or CT imaging was used to assess brain edema. Hematoxylin and eosin staining were used to evaluate cell damage. Immunohistochemistry was used to detect the AQP4 expression. Results: AQP4 expression was increased from 15h to at least 8 d after injury. AQP4immunoreactivity was strong around astrocytomas, ganglioglioma and metastatic adenocarcinoma. However, AQP4 immunoreactivity was only found in the centers of astrocytomas and ganglioglioma, but not in metastatic adenocarcinoma derived from lung.Conclusion: AQP4 expression increases in human brains after traumatic brain injury, within brain-derived tumors, and around brain tumors.

  20. Alternatively spliced short and long isoforms of adaptor protein intersectin 1 form complexes in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rynditch A. V.

    2012-12-01

    Full Text Available Intersectin 1 (ITSN1 is an adaptor protein involved in membrane trafficking and cell signaling. Long and short isoforms of ITSN1 (ITSN1-L and ITSN1-S are produced by alternative splicing. The aim of our study was to investigate whether ITSN1-L and ITSN1-S could interact in mammalian cells. Methods. During this study we employed immunoprecipitation and confocal microscopy. Results. We have shown that endogenous ITSN1-S co-precipitates with overexpressed ITSN1-L in PC12, 293 and 293T cells. Long and short isoforms of ITSN1 also co-localize in 293T cells. Conclusions. ITSN1-L and ITSN1-S form complexes in mammalian cells.

  1. Differential aquaporin 4 expression during edema build-up and resolution phases of brain inflammation

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    Brochet Bruno

    2011-10-01

    Full Text Available Abstract Background Vasogenic edema dynamically accumulates in many brain disorders associated with brain inflammation, with the critical step of edema exacerbation feared in patient care. Water entrance through blood-brain barrier (BBB opening is thought to have a role in edema formation. Nevertheless, the mechanisms of edema resolution remain poorly understood. Because the water channel aquaporin 4 (AQP4 provides an important route for vasogenic edema resolution, we studied the time course of AQP4 expression to better understand its potential effect in countering the exacerbation of vasogenic edema. Methods Focal inflammation was induced in the rat brain by a lysolecithin injection and was evaluated at 1, 3, 7, 14 and 20 days using a combination of in vivo MRI with apparent diffusion coefficient (ADC measurements used as a marker of water content, and molecular and histological approaches for the quantification of AQP4 expression. Markers of active inflammation (macrophages, BBB permeability, and interleukin-1β and markers of scarring (gliosis were also quantified. Results This animal model of brain inflammation demonstrated two phases of edema development: an initial edema build-up phase during active inflammation that peaked after 3 days (ADC increase was followed by an edema resolution phase that lasted from 7 to 20 days post injection (ADC decrease and was accompanied by glial scar formation. A moderate upregulation in AQP4 was observed during the build-up phase, but a much stronger transcriptional and translational level of AQP4 expression was observed during the secondary edema resolution phase. Conclusions We conclude that a time lag in AQP4 expression occurs such that the more significant upregulation was achieved only after a delay period. This change in AQP4 expression appears to act as an important determinant in the exacerbation of edema, considering that AQP4 expression is insufficient to counter the water influx during the build

  2. The diapause program impacts renal excretion and molecular expression of aquaporins in the northern house mosquito, Culex pipiens.

    Science.gov (United States)

    Yang, Liu; Denlinger, David L; Piermarini, Peter M

    2016-12-27

    Adult females of the mosquito Culex pipiens entering diapause increase sugar water ingestion and reduce evaporative water loss, but how these attributes of the diapause program impact activity of the renal excretory system remains unknown. Here we compared the renal excretory capacity of diapausing and non-diapausing females, as well as the molecular expression of aquaporin (AQP) genes that encode channels involved in transporting water and/or small metabolites. Baseline urine excretion rates in diapausing mosquitoes were higher than in those of their non-diapausing counterparts, possibly a consequence of the intense sugar feeding associated with diapause. But, diapausing mosquitoes exhibited a much lower capacity for diuresis than non-diapausing mosquitoes. The suppressed diuretic capacity likely reflects reduced investment in the energetically-expensive post-prandial diuresis, an event not observed in diapausing mosquitoes. The mRNA expression levels of two genes encoding AQPs, Eglp1 and Aqp12L, in diapausing mosquitoes were down-regulated (on day 14) and up-regulated (on both days 3 and 14), respectively, in whole body samples. These changes were not evident in the excretory system (i.e., Malpighian tubules and hindgut), which showed no differential expression of AQPs as a function of diapause. Several AQP mRNAs were, however, differentially expressed in the midgut, ovaries, and abdominal body wall of diapausing mosquitoes, suggesting that AQPs in these tissues may be playing important non-excretory roles that are unique to diapause physiology.

  3. Genome-Wide Identification and Expression Analyses of Aquaporin Gene Family during Development and Abiotic Stress in Banana.

    Science.gov (United States)

    Hu, Wei; Hou, Xiaowan; Huang, Chao; Yan, Yan; Tie, Weiwei; Ding, Zehong; Wei, Yunxie; Liu, Juhua; Miao, Hongxia; Lu, Zhiwei; Li, Meiying; Xu, Biyu; Jin, Zhiqiang

    2015-08-20

    Aquaporins (AQPs) function to selectively control the flow of water and other small molecules through biological membranes, playing crucial roles in various biological processes. However, little information is available on the AQP gene family in bananas. In this study, we identified 47 banana AQP genes based on the banana genome sequence. Evolutionary analysis of AQPs from banana, Arabidopsis, poplar, and rice indicated that banana AQPs (MaAQPs) were clustered into four subfamilies. Conserved motif analysis showed that all banana AQPs contained the typical AQP-like or major intrinsic protein (MIP) domain. Gene structure analysis suggested the majority of MaAQPs had two to four introns with a highly specific number and length for each subfamily. Expression analysis of MaAQP genes during fruit development and postharvest ripening showed that some MaAQP genes exhibited high expression levels during these stages, indicating the involvement of MaAQP genes in banana fruit development and ripening. Additionally, some MaAQP genes showed strong induction after stress treatment and therefore, may represent potential candidates for improving banana resistance to abiotic stress. Taken together, this study identified some excellent tissue-specific, fruit development- and ripening-dependent, and abiotic stress-responsive candidate MaAQP genes, which could lay a solid foundation for genetic improvement of banana cultivars.

  4. Genome-Wide Identification and Expression Analyses of Aquaporin Gene Family during Development and Abiotic Stress in Banana

    Directory of Open Access Journals (Sweden)

    Wei Hu

    2015-08-01

    Full Text Available Aquaporins (AQPs function to selectively control the flow of water and other small molecules through biological membranes, playing crucial roles in various biological processes. However, little information is available on the AQP gene family in bananas. In this study, we identified 47 banana AQP genes based on the banana genome sequence. Evolutionary analysis of AQPs from banana, Arabidopsis, poplar, and rice indicated that banana AQPs (MaAQPs were clustered into four subfamilies. Conserved motif analysis showed that all banana AQPs contained the typical AQP-like or major intrinsic protein (MIP domain. Gene structure analysis suggested the majority of MaAQPs had two to four introns with a highly specific number and length for each subfamily. Expression analysis of MaAQP genes during fruit development and postharvest ripening showed that some MaAQP genes exhibited high expression levels during these stages, indicating the involvement of MaAQP genes in banana fruit development and ripening. Additionally, some MaAQP genes showed strong induction after stress treatment and therefore, may represent potential candidates for improving banana resistance to abiotic stress. Taken together, this study identified some excellent tissue-specific, fruit development- and ripening-dependent, and abiotic stress-responsive candidate MaAQP genes, which could lay a solid foundation for genetic improvement of banana cultivars.

  5. X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking.

    Science.gov (United States)

    Frick, Anna; Eriksson, Urszula Kosinska; de Mattia, Fabrizio; Oberg, Fredrik; Hedfalk, Kristina; Neutze, Richard; de Grip, Willem J; Deen, Peter M T; Törnroth-Horsefield, Susanna

    2014-04-29

    Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein-protein interactions involved in cellular sorting of AQP2. Two Cd(2+)-ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.

  6. The Role of Aquaporins in pH-Dependent Germination of Rhizopus delemar Spores.

    Science.gov (United States)

    Turgeman, Tidhar; Shatil-Cohen, Arava; Moshelion, Menachem; Teper-Bamnolker, Paula; Skory, Christopher D; Lichter, Amnon; Eshel, Dani

    2016-01-01

    Rhizopus delemar and associated species attack a wide range of fruit and vegetables after harvest. Host nutrients and acidic pH are required for optimal germination of R. delemar, and we studied how this process is triggered. Glucose induced spore swelling in an acidic environment, expressed by an up to 3-fold increase in spore diameter, whereas spore diameter was smaller in a neutral environment. When suspended in an acidic environment, the spores started to float, indicating a change in their density. Treatment of the spores with HgCl2, an aquaporin blocker, prevented floating and inhibited spore swelling and germ-tube emergence, indicating the importance of water uptake at the early stages of germination. Two putative candidate aquaporin-encoding genes-RdAQP1 and RdAQP2-were identified in the R. delemar genome. Both presented the conserved NPA motif and six-transmembrane domain topology. Expressing RdAQP1 and RdAQP2 in Arabidopsis protoplasts increased the cells' osmotic water permeability coefficient (Pf) compared to controls, indicating their role as water channels. A decrease in R. delemar aquaporin activity with increasing external pH suggested pH regulation of these proteins. Substitution of two histidine (His) residues, positioned on two loops facing the outer side of the cell, with alanine eliminated the pH sensing resulting in similar Pf values under acidic and basic conditions. Since hydration is critical for spore switching from the resting to activate state, we suggest that pH regulation of the aquaporins can regulate the initial phase of R. delemar spore germination, followed by germ-tube elongation and host-tissue infection.

  7. Maternal-fetal fluid balance and aquaporins: from molecule to physiology

    Institute of Scientific and Technical Information of China (English)

    Xiao-yan SHA; Zheng-fang XIONG; Hui-shu LIU; Xiao-dan DI; Tong-hui MA

    2011-01-01

    Maternal-fetal fluid balance is critical during pregnancy, and amniotic fluid is essential for fetal growth and development. The placenta plays a key role in a successful pregnancy as the interface between the mother and her fetus. Aquaporins (AQPs) form specific water channels that allow the rapid transcellular movement of water in response to osmotic/hydrostatic pressure gradients. AQPs expression in the placenta and fetal membranes may play important roles in the maternal-fetal fluid balance.

  8. Root ABA Accumulation Enhances Rice Seedling Drought Tolerance under Ammonium Supply: Interaction with Aquaporins

    Science.gov (United States)

    Ding, Lei; Li, Yingrui; Wang, Ying; Gao, Limin; Wang, Min; Chaumont, François; Shen, Qirong; Guo, Shiwei

    2016-01-01

    In previous studies, we demonstrated that ammonium nutrition enhances the drought tolerance of rice seedlings compared to nitrate nutrition and contributes to a higher root water uptake ability. It remains unclear why rice seedlings maintain a higher water uptake ability when supplied with ammonium under drought stress. Here, we focused on the effects of nitrogen form and drought stress on root abscisic acid (ABA) concentration and aquaporin expression using hydroponics experiments and stimulating drought stress with 10% PEG6000. Drought stress decreased the leaf photosynthetic rate and stomatal conductivity and increased the leaf temperature of plants supplied with either ammonium or nitrate, but especially under nitrate supply. After 4 h of PEG treatment, the root protoplast water permeability and the expression of root PIP and TIP genes decreased in plants supplied with ammonium or nitrate. After 24 h of PEG treatment, the root hydraulic conductivity, the protoplast water permeability, and the expression of some aquaporin genes increased in plants supplied with ammonium compared to those under non-PEG treatment. Root ABA accumulation was induced by 24 h of PEG treatment, especially in plants supplied with ammonium. The addition of exogenous ABA decreased the expression of PIP and TIP genes under non-PEG treatment but increased the expression of some of them under PEG treatment. We concluded that drought stress induced a down-regulation of aquaporin expression, which appeared earlier than did root ABA accumulation. With continued drought stress, aquaporin expression and activity increased due to root ABA accumulation in plants supplied with ammonium. PMID:27559341

  9. Galactorrhea in a Patient With Aquaporin-4 Antibody-positive Neuromyelitis Optica Spectrum Disorder: A Case Report and Review of the Literature.

    Science.gov (United States)

    Watanabe, Masahiko; Furusho, Kentaro; Takahashi, Toshiyuki; Tamaoka, Akira

    2015-12-01

    This is the first report of a case of galactorrhea in a patient with neuromyelitis optica spectrum disorder (NMOSD) diagnosed on the basis of antiaquaporin-4 antibody seropositivity. The hypothalamus is becoming known as an area highly expressing aquaporin-4 and frequently involved in intracranial lesions of patients with neuromyelitis optica (NMO). We reviewed cases of hypothalamic endocrinopathy among patients with NMO, NMOSD, and the Japanese opticospinal form of MS. Among these cases, galactorrhea was the second most common symptom. Signs of hypothalamic endocrinopathies may be obscured by the grave neurological deficits caused by NMO. We recommend paying special attention to hypothalamic endocrinopathies among patients with NMO or NMOSD, irrespective of brain MRI findings.

  10. Reduction of Skin pH during Treatment for Palmoplantar Hyperhidrosis: A Conjecture on the Role of pH-Regulated Water Channel, i.e. Aquaporin

    Directory of Open Access Journals (Sweden)

    Kyoko Nakahigashi

    2013-04-01

    Full Text Available Primary palmoplantar hyperhidrosis (PPH is a disorder that involves excessive sweating on the palms and soles. Although the pathophysiology of PPH remains unknown, some treatments, including topical aluminum chloride (AC and tap water iontophoresis (TWI, are effective at suppressing the perspiration. Herein, we report the kinetics of the skin pH of two cases of PPH treated with AC and TWI. We found that the skin pH decreased in accordance with the reduction in sweating. This finding indicates that the reduction in sweating may be attributed to the reduction of skin pH in AC and TWI. Whether or not the pH-regulated function of aquaporin can explain this finding remains unknown.

  11. Nonmuscle myosin II isoforms coassemble in living cells.

    Science.gov (United States)

    Beach, Jordan R; Shao, Lin; Remmert, Kirsten; Li, Dong; Betzig, Eric; Hammer, John A

    2014-05-19

    Nonmuscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB, and IIC), each of which possesses distinct biophysical properties and supports unique as well as redundant cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear whether NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments or whether filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently tagged versions of NM IIA, IIB, and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms coassemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while coassembled with other NM II isoforms.

  12. The gating mechanism of the human aquaporin 5 revealed by molecular dynamics simulations.

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    Lorant Janosi

    Full Text Available Aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins. The high-resolution X-ray structure of the human aquaporin 5 (HsAQP5 shows that HsAQP5, as all the other known aquaporins, exhibits tetrameric structure. By means of molecular dynamics simulations we analyzed the role of spontaneous fluctuations on the structural behavior of the human AQP5. We found that different conformations within the tetramer lead to a distribution of monomeric channel structures, which can be characterized as open or closed. The switch between the two states of a channel is a tap-like mechanism at the cytoplasmic end which regulates the water passage through the pore. The channel is closed by a translation of the His67 residue inside the pore. Moreover, water permeation rate calculations revealed that the selectivity filter, located at the other end of the channel, regulates the flow rate of water molecules when the channel is open, by locally modifying the orientation of His173. Furthermore, the calculated permeation rates of a fully open channel are in good agreement with the reported experimental value.

  13. Dynamics and energetics of permeation through aquaporins. What do we learn from molecular dynamics simulations?

    Science.gov (United States)

    Hub, Jochen S; Grubmüller, Helmut; de Groot, Bert L

    2009-01-01

    Aquaporins (AQPs) are a family of integral membrane proteins, which facilitate the rapid and yet highly selective flux of water and other small solutes across biological membranes. Molecular dynamics (MD) simulations contributed substantially to the understanding of the molecular mechanisms that underlie this remarkable efficiency and selectivity of aquaporin channels. This chapter reviews the current state of MD simulations of aquaporins and related aquaglyceroporins as well as the insights these simulations have provided. The mechanism of water permeation through AQPs and methods to determine channel permeabilities from simulations are described. Protons are strictly excluded from AQPs by a large electrostatic barrier and not by an interruption of the Grotthuss mechanism inside the pore. Both the protein's electric field and desolvation effects contribute to this barrier. Permeation of apolar gas molecules such as CO(2) through AQPs is accompanied by a large energetic barrier and thus can only be expected in membranes with a low intrinsic gas permeability. Additionally, the insights from simulations into the mechanism of glycerol permeation through the glycerol facilitator GlpF from E. coli are summarized. Finally, MD simulations are discussed that revealed that the aro-matic/arginine constriction region is generally the filter for uncharged solutes, and that AQP selectivity is controlled by a hydrophobic effect and steric restraints.

  14. NH3 and NH4+ permeability in aquaporin-expressing Xenopus oocytes

    DEFF Research Database (Denmark)

    Holm, Lars M.; Jahn, Thomas Paul; Møller, Anders Laurell Blom;

    2005-01-01

    We have shown recently, in a yeast expression system, that some aquaporins are permeable to ammonia. In the present study, we expressed the mammalian aquaporins AQP8, AQQP9, AQP3, AQP1 and a plant aquaporin TIP2;1 in Xenopus oocytes to study the transport of ammonia (NH3) and ammonium (NH4+) under...... opencircuit and voltage-clamped conditions. TIP2;1 was tested as the wild-type and in a mutated version (tip2;1) in which the water permeability is intact. When AQP8-, AQP9-, AQP3- and TIP2;1-expressing oocytes were placed in a well-stirred bathing medium of low buffer capacity, NH3 permeability was evident...... from the acidification of the bathing medium; the effects observed with AQP1 and tip2;1 did not exceed that of native oocytes. AQP8, AQP9, AQP3, and TIP2;1 were permeable to larger amides, while AQP1 was not. Under voltage-clamp conditions, given sufficient NH3, AQP8, AQP9, AQP3, and TIP2;1 supported...

  15. Increased aquaporin 1 and 5 membrane expression in the lens epithelium of cataract patients.

    Science.gov (United States)

    Barandika, Olatz; Ezquerra-Inchausti, Maitane; Anasagasti, Ander; Vallejo-Illarramendi, Ainara; Llarena, Irantzu; Bascaran, Lucia; Alberdi, Txomin; De Benedetti, Giacomo; Mendicute, Javier; Ruiz-Ederra, Javier

    2016-10-01

    In this work we have analyzed the expression levels of the main aquaporins (AQPs) expressed in human lens epithelial cells (HLECs) using 112 samples from patients treated with cataract surgery and 36 samples from individuals treated with refractive surgery, with transparent lenses as controls. Aquaporin-1 (AQP1) is the main AQP, representing 64.1% of total AQPs in HLECs, with aquaporin-5 (AQP5) representing 35.9% in controls. A similar proportion of each AQP in cataract was found. Although no differences were found at the mRNA level compared to controls, a significant 1.65-fold increase (p=0.001) in AQP1protein expression was observed in HLECs from cataract patients, with the highest differences being found for nuclear cataracts (2.1-fold increase; p<0.001). A similar trend was found for AQP5 (1.47-fold increase), although the difference was not significant (p=0.161). Moreover we have shown increased membrane AQP5 protein expression in HLECs of patients with cataracts. No association of AQP1 or AQP5 expression levels with age or sex was observed in either group. Our results suggest regulation of AQP1 and AQP5 at the post-translational level and support previous observations on the implication of AQP1 and 5 in maintenance of lens transparency in animal models. Our results likely reflect a compensatory response of the crystalline lens to delay cataract formation by increasing the water removal rate.

  16. p53 Family: Role of Protein Isoforms in Human Cancer

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    Jinxiong Wei

    2012-01-01

    Full Text Available TP53, TP63, and TP73 genes comprise the p53 family. Each gene produces protein isoforms through multiple mechanisms including extensive alternative mRNA splicing. Accumulating evidence shows that these isoforms play a critical role in the regulation of many biological processes in normal cells. Their abnormal expression contributes to tumorigenesis and has a profound effect on tumor response to curative therapy. This paper is an overview of isoform diversity in the p53 family and its role in cancer.

  17. The Prevalence of Anti-Aquaporin 4 Antibody in Patients with Idiopathic Inflammatory Demyelinating Diseases Presented to a Tertiary Hospital in Malaysia: Presentation and Prognosis

    Science.gov (United States)

    Tan, C. T.

    2017-01-01

    Background. There have been inconsistent reports on the prevalence and pathogenicity of anti-Aquaporin 4 (AQP4) in patients presented with idiopathic inflammatory demyelinating diseases (IIDDs). Objective. To estimate the prevalence of anti-AQP4 antibody in patients with IIDDs presented to University Malaya Medical Centre in terms of patients' clinical and radiological presentations and prognoses. Methods. Retrospective data review of IIDDs patients presented from 2005 to 2015. Patients were classified into classical multiple sclerosis (CMS), opticospinal (OS) presentation, optic neuritis (ON), transverse myelitis (TM), brainstem syndrome (BS), and tumefactive MS. Anti-Aquaporin 4 antibody was tested using the Indirect Immunofluorescence Test (IIFT) cell-based assay. Statistical analysis was done using the SPSS version 20. Results. Anti-AQP4 antibody was detected in 53% of patients presented with IIDDs. CMS was more common in the seronegative group, 27/47 (57.45%; p < 0.001). Conversely, OS involvement was more common in the seropositive group, 26/53 (49.06%; p < 0.001). Longitudinally extensive spinal cord lesions (LESCLs) on MRI were also more common in the seropositive group, 29/40 (72.50%; p = 0.004). Only 2/40 (5.00%) had MRI evidence of patchy or multiple short-segment spinal cord lesions in the AQP4-positive group (p = 0.003). The relapse rate and Expanded Disability Status Scale (EDSS) were also higher in the seropositive group (5.43 versus 3.17, p = 0.005; 4.07 versus 2.51, p = 0.006, resp.). Typical clinical presentations that defined NMO were also seen in the seronegative patients, but in a lower frequency. Conclusion. Our cohort of patients had a higher prevalence of seropositivity of anti-AQP4 antibody as compared to those in Western countries. This was also associated with a more typical presentation of opticospinal involvement with LESCLs on MRI, a higher rate of relapse, and EDSS. PMID:28203460

  18. Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer.

    Science.gov (United States)

    Yang, Ting; Xu, Feifei; Fang, Danjun; Chen, Yun

    2015-11-17

    The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed.

  19. Conditional expression of CD44 isoforms in lymphoma cells: influence on hyaluronate binding and tumor growth

    Energy Technology Data Exchange (ETDEWEB)

    Fu, J.

    2002-03-01

    CD44 describes a family of surface proteins consisting of many isoforms due to alternative splice of ten 'variant' exons. Members of this family are involved in various processes including hematopoiesis, lymphocyte activation and homing, limb development, wound healing and tumor progression. Clinically, CD44 has been shown to be a prognostic factor for several human cancers. To answer the question which isoform might be relevant for tumor progression and to gain an insight into the mechanism of its function, I established transfectants of the LB lymphoma cell line in which the expression of four CD44 isoforms, namely CD44v3-10, CD44v4-10, CD44v8-10 and CD44s, was controlled by the Tet-off promoter. In the presence of Doxycycline, the expression was repressed. Removal of Doxycycline switched on expression and the maximal CD44 amount was obtained within two days. The transfectants were characterized regarding their ability to bind to the extracellular matrix component hyaluronate (HA). Overexpression of all four CD44 isoforms conferred the ability to bind HA on LB cells. Other glycosaminoglycans (GAGs) were bound in an isotype-specific fashion. CD44v3-10, CD44v4-10 and CD44v8-10 showed high binding affinity to chondroitin A, B and C, and low affinity to heparin, heparan sulfate and keratan sulfate. CD44s could not bind to these GAGs. Among these three variants, the binding ability of CD44v3-10 was the strongest. CD44 clustering seemed to play a crucial role for HA binding. Both CD44s and CD44v8-10 formed reduction-sensitive complexes in LB cells. The complexes are homooligomers or heterooligomers composed of different isoforms. Cys286 in CD44 transmember domain was not responsible for the formation of reduction-sensitive oligomer or for the enhanced HA binding in LB cell line. Using a conditional dimerization system the requirement of CD44 oligomerization for HA binding was directly demonstrated. The induction of oligomerization increased HA binding

  20. Temporal patterns of broad isoform expression during the development of neuronal lineages in Drosophila

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    Williams Darren W

    2009-11-01

    Full Text Available Abstract Background During the development of the central nervous system (CNS of Drosophila, neuronal stem cells, the neuroblasts (NBs, first generate a set of highly diverse neurons, the primary neurons that mature to control larval behavior, and then more homogeneous sets of neurons that show delayed maturation and are primarily used in the adult. These latter, 'secondary' neurons show a complex pattern of expression of broad, which encodes a transcription factor usually associated with metamorphosis, where it acts as a key regulator in the transitions from larva and pupa. Results The Broad-Z3 (Br-Z3 isoform appears transiently in most central neurons during embryogenesis, but persists in a subset of these cells through most of larval growth. Some of the latter are embryonic-born secondary neurons, whose development is arrested until the start of metamorphosis. However, the vast bulk of the secondary neurons are generated during larval growth and bromodeoxyuridine incorporation shows that they begin expressing Br-Z3 about 7 hours after their birth, approximately the time that they have finished outgrowth to their initial targets. By the start of metamorphosis, the oldest secondary neurons have turned off Br-Z3 expression, while the remainder, with the exception of the very youngest, maintain Br-Z3 while they are interacting with potential partners in preparation for neurite elaboration. That Br-Z3 may be involved in early sprouting is suggested by ectopically expressing this isoform in remodeling primary neurons, which do not normally express Br-Z3. These cells now sprout into ectopic locations. The expression of Br-Z3 is transient and seen in all interneurons, but two other isoforms, Br-Z4 and Br-Z1, show a more selective expression. Analysis of MARCM clones shows that the Br-Z4 isoform is expressed by neurons in virtually all lineages, but only in those cells born during a window during the transition from the second to the third larval

  1. Neuronal profilin isoforms are addressed by different signalling pathways.

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    Kai Murk

    Full Text Available Profilins are prominent regulators of actin dynamics. While most mammalian cells express only one profilin, two isoforms, PFN1 and PFN2a are present in the CNS. To challenge the hypothesis that the expression of two profilin isoforms is linked to the complex shape of neurons and to the activity-dependent structural plasticity, we analysed how PFN1 and PFN2a respond to changes of neuronal activity. Simultaneous labelling of rodent embryonic neurons with isoform-specific monoclonal antibodies revealed both isoforms in the same synapse. Immunoelectron microscopy on brain sections demonstrated both profilins in synapses of the mature rodent cortex, hippocampus and cerebellum. Both isoforms were significantly more abundant in postsynaptic than in presynaptic structures. Immunofluorescence showed PFN2a associated with gephyrin clusters of the postsynaptic active zone in inhibitory synapses of embryonic neurons. When cultures were stimulated in order to change their activity level, active synapses that were identified by the uptake of synaptotagmin antibodies, displayed significantly higher amounts of both isoforms than non-stimulated controls. Specific inhibition of NMDA receptors by the antagonist APV in cultured rat hippocampal neurons resulted in a decrease of PFN2a but left PFN1 unaffected. Stimulation by the brain derived neurotrophic factor (BDNF, on the other hand, led to a significant increase in both synaptic PFN1 and PFN2a. Analogous results were obtained for neuronal nuclei: both isoforms were localized in the same nucleus, and their levels rose significantly in response to KCl stimulation, whereas BDNF caused here a higher increase in PFN1 than in PFN2a. Our results strongly support the notion of an isoform specific role for profilins as regulators of actin dynamics in different signalling pathways, in excitatory as well as in inhibitory synapses. Furthermore, they suggest a functional role for both profilins in neuronal nuclei.

  2. Isoform-specific targeting of ROCK proteins in immune cells

    OpenAIRE

    Zanin-Zhorov, Alexandra; Flynn, Ryan; Waksal, Samuel D.; Blazar, Bruce R.

    2016-01-01

    ABSTRACT Rho-associated kinase 1 (ROCK1) and ROCK2 are activated by Rho GTPase and control cytoskeleton rearrangement through modulating the phosphorylation of their down-stream effector molecules. Although these 2 isoforms share more than 90% homology within their kinase domain the question of whether ROCK proteins function identically in different cell types is not clear. By using both pharmacological inhibition and genetic knockdown approaches recent studies suggest that the ROCK2 isoform ...

  3. Genome-wide identification of aquaporin encoding genes in Brassica oleracea and their phylogenetic sequence comparison to Brassica crops and Arabidopsis

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    Till Arvid Diehn

    2015-04-01

    Full Text Available Aquaporins (AQPs are essential channel proteins that regulate plant water homeostasis and the uptake and distribution of uncharged solutes such as metalloids, urea, ammonia and carbon dioxide. Despite their importance as crop plants, little is known about AQP gene and protein function in cabbage (Brassica oleracea and other Brassica species. The recent releases of the genome sequences of B. oleracea and B. rapa allow comparative genomic studies in these species to investigate the evolution and features of Brassica genes and proteins.In this study, we identified all AQP genes in B. oleracea by a genome-wide survey. In total, 67 genes of four plant AQP subfamilies were identified. Their full-length gene sequences and locations on chromosomes and scaffolds were manually curated. The identification of six additional full-length AQP sequences in the B. rapa genome added to the recently published AQP protein family of this species. A phylogenetic analysis of AQPs of A. thaliana, B. oleracea, B. rapa allowed us to follow AQP evolution in closely related species and to systematically classify and (re- name these isoforms. Thirty-three groups of AQP-orthologous genes were identified between B. oleracea and Arabidopsis and their expression was analyzed in different organs. The two selectivity filters, gene structure and coding sequences were highly conserved within each AQP subfamily while sequence variations in some introns and untranslated regions were frequent. These data suggest a similar substrate selectivity and function of Brassica AQPs compared to Arabidopsis orthologs. The comparative analyses of all AQP subfamilies in three Brassicaceae species give initial insights into AQP evolution in these taxa. Based on the genome-wide AQP identification in B. oleracea and the sequence analysis and reprocessing of Brassica AQP information, our dataset provides a sequence resource for further investigations of the physiological and molecular functions of

  4. Genome-wide identification of aquaporin encoding genes in Brassica oleracea and their phylogenetic sequence comparison to Brassica crops and Arabidopsis.

    Science.gov (United States)

    Diehn, Till A; Pommerrenig, Benjamin; Bernhardt, Nadine; Hartmann, Anja; Bienert, Gerd P

    2015-01-01

    Aquaporins (AQPs) are essential channel proteins that regulate plant water homeostasis and the uptake and distribution of uncharged solutes such as metalloids, urea, ammonia, and carbon dioxide. Despite their importance as crop plants, little is known about AQP gene and protein function in cabbage (Brassica oleracea) and other Brassica species. The recent releases of the genome sequences of B. oleracea and Brassica rapa allow comparative genomic studies in these species to investigate the evolution and features of Brassica genes and proteins. In this study, we identified all AQP genes in B. oleracea by a genome-wide survey. In total, 67 genes of four plant AQP subfamilies were identified. Their full-length gene sequences and locations on chromosomes and scaffolds were manually curated. The identification of six additional full-length AQP sequences in the B. rapa genome added to the recently published AQP protein family of this species. A phylogenetic analysis of AQPs of Arabidopsis thaliana, B. oleracea, B. rapa allowed us to follow AQP evolution in closely related species and to systematically classify and (re-) name these isoforms. Thirty-three groups of AQP-orthologous genes were identified between B. oleracea and Arabidopsis and their expression was analyzed in different organs. The two selectivity filters, gene structure and coding sequences were highly conserved within each AQP subfamily while sequence variations in some introns and untranslated regions were frequent. These data suggest a similar substrate selectivity and function of Brassica AQPs compared to Arabidopsis orthologs. The comparative analyses of all AQP subfamilies in three Brassicaceae species give initial insights into AQP evolution in these taxa. Based on the genome-wide AQP identification in B. oleracea and the sequence analysis and reprocessing of Brassica AQP information, our dataset provides a sequence resource for further investigations of the physiological and molecular functions of

  5. Cell-specific expression of TLR9 isoforms in inflammation.

    Science.gov (United States)

    McKelvey, Kelly J; Highton, John; Hessian, Paul A

    2011-02-01

    Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

  6. Novel isoform of adaptor protein ITSN1 forms homodimers via its C-terminus

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    Skrypkina I. Ya.

    2011-06-01

    Full Text Available Aim. Previously we have identified a novel isoform of endocytic adaptor protein ITSN1 designated as ITSN122a. Western blot revealed two immunoreactive bands of 120 and 250 kDa that corresponded to ITSN1-22a. The goal of this study was to investigate the possibility of dimer formation by the novel isoform. Methods. Dimerization ability of ITSN1-22a was tested by immunoprecipitation and subsequent Western blot analysis. To specify the region responsible for dimerization, site-directed mutagenesis and truncation analysis were carried out. Inhibition of endocytosis by potassium depletion and EGF stimulation of HEK293 were performed. Results. We have found that ITSN1-22a forms dimers in HEK293 cells. The dimerization of ITSN1-22a was mediated by C-terminal domain. We showed that cysteines C1016 and C1019 were involved in homodimerization. Inhibition of clathrin-mediated endocytosis and mitogen stimulation did not affect ITSN1-22a dimer formation. Conclusions. ITSN1-22a is the only one known ITSN1 isoform, which is capable to form homodimers via disulphide bonds. This could be important for the formation of protein complexes containing ITSN1 molecules.

  7. Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent.

    Science.gov (United States)

    Kim, Hak Rim; Gallant, Cynthia; Leavis, Paul C; Gunst, Susan J; Morgan, Kathleen G

    2008-09-01

    Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an alpha-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses alpha-smooth muscle actin, beta-actin, nonmuscle gamma-actin, and smooth muscle gamma-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during alpha-agonist contractions involves the remodeling of primarily gamma-actin and, to a lesser extent, beta-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

  8. Fruitless isoforms and target genes specify the sexually dimorphic nervous system underlying Drosophila reproductive behavior.

    Science.gov (United States)

    Nojima, Tetsuya; Neville, Megan C; Goodwin, Stephen F

    2014-01-01

    Courtship is pivotal to successful reproduction throughout the animal kingdom. Sexual differences in the nervous system are thought to underlie courtship behavior. Male courtship behavior in Drosophila is in large part regulated by the gene fruitless (fru). fru has been reported to encode at least three putative BTB-zinc-finger transcription factors predicted to have different DNA-binding specificities. Although a large number of previous studies have demonstrated that fru plays essential roles in male courtship behavior, we know little about the function of Fru isoforms at the molecular level. Our recent study revealed that male-specific Fru isoforms are expressed in highly overlapping subsets of neurons in the male brain and ventral nerve cord. Fru isoforms play both distinct and redundant roles in male courtship behavior. Importantly, we have identified for the first time, by means of the DamID technique, direct Fru transcriptional target genes. Fru target genes overwhelmingly represent genes previously reported to be involved in the nervous system development, such as CadN, lola and pdm2. Our study provides important insight into how the sexually dimorphic neural circuits underlying reproductive behavior are established.

  9. Mechanisms controlling Pax6 isoform expression in the retina have been conserved between teleosts and mammals.

    Science.gov (United States)

    Lakowski, Jörn; Majumder, Anirban; Lauderdale, James D

    2007-07-15

    The Pax6 gene plays several roles in retinal development, including control of cell proliferation, maintenance of the retinogenic potential of progenitor cells, and cell fate specification. Emerging evidence suggests that these different aspects of Pax6 gene function are mediated by different isoforms of the Pax6 protein; however, relatively little is known about the spatiotemporal expression of Pax6 isoforms in the vertebrate retina. Using bacterial artificial chromosome (BAC) technology, we modified a zebrafish Pax6a BAC such that we could distinguish paired-containing Pax6a transcripts from paired-less Pax6a transcripts. In the zebrafish, the spatial and temporal onset of expression of these transcripts suggests that the paired-less isoform is involved in the cell fate decision leading to the generation of amacrine cells; however, because of limitations associated with transient transgenic analysis, it was not feasible to establish whether this promoter was active in all amacrine cells or in a specific population of amacrine cells. By making mice transgenic for the zebrafish Pax6a BAC reporter transgene, we were able to show that paired-containing and paired-less Pax6a transcripts were differentially expressed in amacrine subpopulations. Our study also directly demonstrates the functional conservation of the regulatory mechanisms governing Pax6 transcription in teleosts and mammals.

  10. Recombinant erythropoietin in humans has a prolonged effect on circulating erythropoietin isoform distribution

    DEFF Research Database (Denmark)

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian

    2014-01-01

    The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross...

  11. Role of JNK isoforms in the development of neuropathic pain following sciatic nerve transection in the mouse

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    Manassero Giusi

    2012-05-01

    Full Text Available Abstract Background Current tools for analgesia are often only partially successful, thus investigations of new targets for pain therapy stimulate great interest. Consequent to peripheral nerve injury, c-Jun N-terminal kinase (JNK activity in cells of the dorsal root ganglia (DRGs and spinal cord is involved in triggering neuropathic pain. However, the relative contribution of distinct JNK isoforms is unclear. Using knockout mice for single isoforms, and blockade of JNK activity by a peptide inhibitor, we have used behavioral tests to analyze the contribution of JNK in the development of neuropathic pain after unilateral sciatic nerve transection. In addition, immunohistochemical labelling for the growth associated protein (GAP-43 and Calcitonin Gene Related Peptide (CGRP in DRGs was used to relate injury related compensatory growth to altered sensory function. Results Peripheral nerve injury produced pain–related behavior on the ipsilateral hindpaw, accompanied by an increase in the percentage of GAP43-immunoreactive (IR neurons and a decrease in the percentage of CGRP-IR neurons in the lumbar DRGs. The JNK inhibitor, D-JNKI-1, successfully modulated the effects of the sciatic nerve transection. The onset of neuropathic pain was not prevented by the deletion of a single JNK isoform, leading us to conclude that all JNK isoforms collectively contribute to maintain neuropathy. Autotomy behavior, typically induced by sciatic nerve axotomy, was absent in both the JNK1 and JNK3 knockout mice. Conclusions JNK signaling plays an important role in regulating pain threshold: the inhibition of all of the JNK isoforms prevents the onset of neuropathic pain, while the deletion of a single splice JNK isoform mitigates established sensory abnormalities. JNK inactivation also has an effect on axonal sprouting following peripheral nerve injury.

  12. Multiple isoform recovery (MIR)-PCR: a simple method for the isolation of related mRNA isoforms.

    OpenAIRE

    Fagotti, A; Gabbiani, G.; Pascolini, R; Neuville, P

    1998-01-01

    We present a rapid and efficient method for the detection of related transcripts with different expression levels. This approach combines the rapid amplification of cDNA ends (RACE) method with a cDNA subtractive technique. The strategy is based on successive subtractions of prevalent isoforms resulting in enrichment of less expressed transcripts. For each subtraction, a biotinylated primer specific for the prevalent isoform is hybridized on the total cDNA and the hybrid is retained on a stre...

  13. The aromatic/arginine selectivity filter of NIP aquaporins plays a critical role in substrate selectivity for silicon, boron, and arsenic.

    Science.gov (United States)

    Mitani-Ueno, Namiki; Yamaji, Naoki; Zhao, Fang-Jie; Ma, Jian Feng

    2011-08-01

    Nodulin-26-like intrinsic proteins (NIPs) of the aquaporin family are involved in the transport of diverse solutes, but the mechanisms controlling the selectivity of transport substrates are poorly understood. The purpose of this study was to investigate how the aromatic/arginine (ar/R) selectivity filter influences the substrate selectivity of two NIP aquaporins; the silicic acid (Si) transporter OsLsi1 (OsNIP2;1) from rice and the boric acid (B) transporter AtNIP5;1 from Arabidopsis; both proteins are also permeable to arsenite. Native and site-directed mutagenized variants of the two genes were expressed in Xenopus oocytes and the transport activities for Si, B, arsenite, and water were assayed. Substitution of the amino acid at the ar/R second helix (H2) position of OsLsi1 did not affect the transport activities for Si, B, and arsenite, but that at the H5 position resulted in a total loss of Si and B transport activities and a partial loss of arsenite transport activity. Conversely, changes of the AtNIP5;1 ar/R selectivity filter and the NPA motifs to the OsLsi1 type did not result in a gain of Si transport activity. B transport activity was partially lost in the H5 mutant but unaffected in the H2 mutant of AtNIP5;1. In contrast, both the single and double mutations at the H2 and/or H5 positions of AtNIP5;1 did not affect arsenite transport activity. The results reveal that the residue at the H5 position of the ar/R filter of both OsLsi1 and AtNIP5;1 plays a key role in the permeability to Si and B, but there is a relatively low selectivity for arsenite.

  14. Pollen-Specific Aquaporins NIP4;1 and NIP4;2 Are Required for Pollen Development and Pollination in Arabidopsis thaliana.

    Science.gov (United States)

    Di Giorgio, Juliana Andrea Pérez; Bienert, Gerd Patrick; Ayub, Nicolás Daniel; Yaneff, Agustín; Barberini, María Laura; Mecchia, Martín Alejandro; Amodeo, Gabriela; Soto, Gabriela Cynthia; Muschietti, Jorge Prometeo

    2016-05-01

    In flowers with dry stigmas, pollen development, pollination, and pollen tube growth require spatial and temporal regulation of water and nutrient transport. To better understand the molecular mechanisms involved in reproductive processes, we characterized NIP4;1 and NIP4;2, two pollen-specific aquaporins of Arabidopsis thaliana. NIP4;1 and NIP4;2 are paralogs found exclusively in the angiosperm lineage. Although they have 84% amino acid identity, they displayed different expression patterns. NIP4;1 has low expression levels in mature pollen, while NIP4;2 expression peaks during pollen tube growth. Additionally, NIP4;1pro:GUS flowers showed GUS activity in mature pollen and pollen tubes, whereas NIP4;2pro:GUS flowers only in pollen tubes. Single T-DNA mutants and double artificial microRNA knockdowns had fewer seeds per silique and reduced pollen germination and pollen tube length. Transport assays in oocytes showed NIP4;1 and NIP4;2 function as water and nonionic channels. We also found that NIP4;1 and NIP4;2 C termini are phosphorylated by a pollen-specific CPK that modifies their water permeability. Survival assays in yeast indicated that NIP4;1 also transports ammonia, urea, boric acid, and H2O2 Thus, we propose that aquaporins NIP4;1 and NIP4;2 are exclusive components of the reproductive apparatus of angiosperms with partially redundant roles in pollen development and pollination.

  15. The role of aquaporin and tight junction proteins in the regulation of water movement in larval zebrafish (Danio rerio.

    Directory of Open Access Journals (Sweden)

    Raymond W M Kwong

    Full Text Available Teleost fish living in freshwater are challenged by passive water influx; however the molecular mechanisms regulating water influx in fish are not well understood. The potential involvement of aquaporins (AQP and epithelial tight junction proteins in the regulation of transcellular and paracellular water movement was investigated in larval zebrafish (Danio rerio. We observed that the half-time for saturation of water influx (K(u was 4.3±0.9 min, and reached equilibrium at approximately 30 min. These findings suggest a high turnover rate of water between the fish and the environment. Water influx was reduced by the putative AQP inhibitor phloretin (100 or 500 μM. Immunohistochemistry and confocal microscopy revealed that AQP1a1 protein was expressed in cells on the yolk sac epithelium. A substantial number of these AQP1a1-positive cells were identified as ionocytes, either H⁺-ATPase-rich cells or Na⁺/K⁺-ATPase-rich cells. AQP1a1 appeared to be expressed predominantly on the basolateral membranes of ionocytes, suggesting its potential involvement in regulating ionocyte volume and/or water flux into the circulation. Additionally, translational gene knockdown of AQP1a1 protein reduced water influx by approximately 30%, further indicating a role for AQP1a1 in facilitating transcellular water uptake. On the other hand, incubation with the Ca²⁺-chelator EDTA or knockdown of the epithelial tight junction protein claudin-b significantly increased water influx. These findings indicate that the epithelial tight junctions normally act to restrict paracellular water influx. Together, the results of the present study provide direct in vivo evidence that water movement can occur through transcellular routes (via AQP; the paracellular routes may become significant when the paracellular permeability is increased.

  16. Development of supported biomimetic membranes for insertion of aquaporin protein water channels for novel water filtration applications

    DEFF Research Database (Denmark)

    Hansen, Jesper Søndergaard

    to develop novel water separation technologies. To accomplish this, it is necessary to construct an efficient platform to handle biomimetic membranes. Moreover, general methods are required to reliable and controllable reconstitute membrane proteins into artificially made model membranes......Aquaporins represent a class of membrane protein channels found in all living organisms that selectively transport water molecules across biological membranes. The work presented in this thesis was motivated by the conceptual idea of incorporating aquaporin water channels into biomimetic membranes......). This constitutes a new methodology to correctly and functionally reconstitute membrane proteins in controllable amounts into giant vesicles. The method for formation of giant protein vesicles subsequently led to the first functional prototype of an aquaporin-membrane water filtration device....

  17. The differences in binding 12-carbon aliphatic ligands by bovine β-lactoglobulin isoform A and B studied by isothermal titration calorimetry and X-ray crystallography.

    Science.gov (United States)

    Loch, Joanna I; Bonarek, Piotr; Polit, Agnieszka; Swiątek, Śylwia; Dziedzicka-Wasylewska, Marta; Lewiński, Krzysztof

    2013-08-01

    Isoforms A (LGB-A) and B (LGB-B) of bovine lactoglobulin, the milk protein, differ in positions 64 (D↔G) and 118 (V↔A). Interactions of LGB-A and LGB-B with sodium dodecyl sulfate (SDS), dodecyltrimethylammonium chloride (DTAC) and lauric acid (LA), 12-carbon ligands possessing differently charged polar groups, were investigated using isothermal titration calorimetry and X-ray crystallography, to study the proton linkage phenomenon and to distinguish between effects related to different isoforms and different ligand properties. The determined values of ΔS and ΔH revealed that for all ligands, binding is entropically driven. The contribution from enthalpy change is lower and shows strong dependence on type of buffer that indicates proton release from the protein varying with protein isoform and ligand type and involvement of LA and Asp64 (in isoform A) in this process. The ligand affinities for both isoforms were arranged in the same order, DTAC enthalpy, resulting in almost identical ΔG for complexes of both isoforms. The determined crystal structures showed that substitution in positions 64 and 118 did not influence the overall structure of LGB complexes. The chemical character of the ligand polar group did not affect the position of its aliphatic chain in protein β-barrel, indicating a major role of hydrophobic interactions in ligand binding that prevailed even with the repulsion between positively charged DTAC and lysine residues located at binding site entrance.

  18. Differential activities of glucocorticoid-induced leucine zipper protein isoforms.

    Science.gov (United States)

    Soundararajan, Rama; Wang, Jian; Melters, Daniël; Pearce, David

    2007-12-14

    Glucocorticoid-induced leucine zipper protein (GILZ) is expressed in both epithelial and immune tissues and modulates a variety of cellular functions, including proliferation and epithelial sodium channel (ENaC) activity. A number of reports have described various GILZ activities, focusing on a single isoform with molecular mass of approximately 17 kDa, now termed GILZ1. In GILZ immunoblots using a newly developed antiserum, we detected multiple species in extracts from cultured kidney cells. Mass spectrometric analysis revealed that one of these represented a previously uncharacterized distinct isoform of GILZ, GILZ2. Rapid amplification of cDNA ends was used to clone cDNAs corresponding to four isoforms, which, in addition to GILZ1 and GILZ2, included new isoforms GILZ3 and GILZ4. Heterologous expression of these four GILZ isoforms in cultured cells revealed striking functional differences. Notably, GILZ1 was the only isoform that significantly stimulated ENaC-mediated Na+ current in a kidney collecting duct cell line, although GILZ2 and GILZ3 also stimulated ENaC surface expression in HEK 293 cells. GILZ1 and GILZ3, and to a lesser extent GILZ2, inhibited ERK phosphorylation. Interestingly, GILZ4, which had no effect on either ENaC or ERK, potently suppressed cellular proliferation, as did GILZ1, but not GILZ2 or GILZ3. Finally, rat and mouse tissues all expressed multiple GILZ species but varied in the relative abundance of each. These data suggest that multiple GILZ isoforms are expressed in most cells and tissues and that these play distinct roles in regulating key cellular functions, including proliferation and ion transport. Furthermore, GILZ inhibition of ERK appears to play an essential role in stimulation of cell surface ENaC but not in inhibition of proliferation.

  19. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

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    Mirco Müller

    Full Text Available Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

  20. GFAP isoforms in adult mouse brain with a focus on neurogenic astrocytes and reactive astrogliosis in mouse models of Alzheimer disease.

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    Willem Kamphuis

    Full Text Available Glial fibrillary acidic protein (GFAP is the main astrocytic intermediate filament (IF. GFAP splice isoforms show differential expression patterns in the human brain. GFAPδ is preferentially expressed by neurogenic astrocytes in the subventricular zone (SVZ, whereas GFAP(+1 is found in a subset of astrocytes throughout the brain. In addition, the expression of these isoforms in human brain material of epilepsy, Alzheimer and glioma patients has been reported. Here, for the first time, we present a comprehensive study of GFAP isoform expression in both wild-type and Alzheimer Disease (AD mouse models. In cortex, cerebellum, and striatum of wild-type mice, transcripts for Gfap-α, Gfap-β, Gfap-γ, Gfap-δ, Gfap-κ, and a newly identified isoform Gfap-ζ, were detected. Their relative expression levels were similar in all regions studied. GFAPα showed a widespread expression whilst GFAPδ distribution was prominent in the SVZ, rostral migratory stream (RMS, neurogenic astrocytes of the subgranular zone (SGZ, and subpial astrocytes. In contrast to the human SVZ, we could not establish an unambiguous GFAPδ localization in proliferating cells of the mouse SVZ. In APPswePS1dE9 and 3xTgAD mice, plaque-associated reactive astrocytes had increased transcript levels of all detectable GFAP isoforms and low levels of a new GFAP isoform, Gfap-ΔEx7. Reactive astrocytes in AD mice showed enhanced GFAPα and GFAPδ immunolabeling, less frequently increased vimentin and nestin, but no GFAPκ or GFAP(+1 staining. In conclusion, GFAPδ protein is present in SVZ, RMS, and neurogenic astrocytes of the SGZ, but also outside neurogenic niches. Furthermore, differential GFAP isoform expression is not linked with aging or reactive gliosis. This evidence points to the conclusion that differential regulation of GFAP isoforms is not involved in the reorganization of the IF network in reactive gliosis or in neurogenesis in the mouse brain.

  1. Different expression patterns of renal Na(+)/K(+)-ATPase α-isoform-like proteins between tilapia and milkfish following salinity challenges.

    Science.gov (United States)

    Yang, Wen-Kai; Chung, Chang-Hung; Cheng, Hui Chen; Tang, Cheng-Hao; Lee, Tsung-Han

    2016-12-01

    Euryhaline teleosts can survive in a broad range of salinity via alteration of the molecular mechanisms in certain osmoregulatory organs, including in the gill and kidney. Among these mechanisms, Na(+)/K(+)-ATPase (NKA) plays a crucial role in triggering ion-transporting systems. The switch of NKA isoforms in euryhaline fish gills substantially contributes to salinity adaptation. However, there is little information about switches in the kidneys of euryhaline teleosts. Therefore, the responses of the renal NKA α-isoform protein switch to salinity challenge in euryhaline tilapia (Oreochromis mossambicus) and milkfish (Chanos chanos) with different salinity preferences were examined and compared in this study. Immunohistochemical staining in tilapia kidneys revealed the localization of NKA in renal tubules rather than in the glomeruli, similar to our previous findings in milkfish kidneys. Protein abundance in the renal NKA pan α-subunit-like, α1-, and α3-isoform-like proteins in seawater-acclimated tilapia was significantly higher than in the freshwater group, whereas the α2-isoform-like protein exhibited the opposite pattern of expression. In the milkfish, higher protein abundance in the renal NKA pan α-subunit-like and α1-isoform-like proteins was found in freshwater-acclimated fish, whereas no difference was found in the protein abundance of α2- and α3-isoform-like proteins between groups. These findings suggested that switches for renal NKA α-isoforms, especially the α1-isoform, were involved in renal osmoregulatory mechanisms of euryhaline teleosts. Moreover, differences in regulatory responses of the renal NKA α-subunit to salinity acclimation between tilapia and milkfish revealed that divergent mechanisms for maintaining osmotic balance might be employed by euryhaline teleosts with different salinity preferences.

  2. Detection of aquaporin-4 antibody using aquaporin-4 extracellular loop-based carbon nanotube biosensor for the diagnosis of neuromyelitis optica.

    Science.gov (United States)

    Son, Manki; Kim, Daesan; Park, Kyung Seok; Hong, Seunghun; Park, Tai Hyun

    2016-04-15

    Here we propose a carbon nanotube (CNT) field-effect transistor (FET) functionalized with aquaporin-4 (AQP4) extracellular loop peptides for the rapid detection of AQP4 antibody without pretreatment. Neuromyelitis optica (NMO) is a rare disease of the central nerve system that affects the optic nerves and the spinal cord. NMO-IgG, a serum antibody in patients, is highly specific for NMO and targets AQP4. We synthesized AQP4 extracellular loop peptides, known as primary autoimmune target in NMO, and immobilized them onto CNT-FET. The sensor showed p-type FET characteristics after the functionalization of peptides. The sensor was able to detect antibody with a detection limit of 1 ng l(-1). Moreover, AQP4 antibody in human serum was detected without any pretreatment. These results indicate that the biosensor can be used for rapid and simple detection of NMO antibody.

  3. Isoform-targeted regulation of cardiac adenylyl cyclase.

    Science.gov (United States)

    Ishikawa, Yoshihiro

    2003-01-01

    Numerous attempts have been made to develop strategies for regulating the intracellular cyclic AMP signal pharmacologically, with an intention to establish either new medical therapeutic methods or experimental tools. In the past decades, many pharmacological reagents have been identified that regulate this pathway at the level of the receptor. G protein, adenylyl cyclase, cyclic AMP, protein kinase A and phosphodiesterase. Since the cloning of adenylyl cyclase isoforms during the 1990s, investigators including ourselves have tried to find reagents that regulate the activity of this enzyme directly in an isoform-dependent manner. The ultimate goal of developing such reagents would be to regulate the cyclic AMP signal in an organ-dependent manner. Ourselves and other workers have reported that such reagents may vary from a simple cation to kinases. In a more recent study, using the results from crystallographic studies and computer-assisted drug design programs, we have identified subtype-selective regulators of adenylyl cyclase. Such regulators are mostly based upon forskolin, a diterpene compound obtained from Coleus forskolii, that acts directly on adenylyl cyclase to increase the intracellular levels of cyclic AMP. Similarly, novel reagents have been identified that inhibit a specific adenylyl cyclase isoform (e.g. type 5 adenylyl cyclase). Such reagents would potentially provide a new therapeutic strategy to treat hypertension, for example, as well as methods to selectively stimulate or inhibit this adenylyl cyclase isoform, which may be reminiscent of overexpression or knocking out of the cardiac adenylyl cyclase isoform by the use of a pharmacological method.

  4. Downregulation of aquaporin-1 in alveolar microvessels in lungs adapted to chronic heart failure

    DEFF Research Database (Denmark)

    Müllertz, Katrine M; Strøm, Claes; Trautner, Simon

    2011-01-01

    The threshold pressure for lung edema formation is increased in severe chronic heart failure (CHF) due to reduced microvascular permeability. The water channel aquaporin-1 (AQP1) is present in the pulmonary microvascular endothelium, and a number of studies suggest the importance of AQP1...... as a compensatory mechanism to protect against formation of excessive pulmonary edema in CHF....... as a molecular determinant of pulmonary microvascular water transport. The present study examined the abundance and localization of AQP1 in lungs from rats with CHF. We used two different models of CHF: ligation of the left anterior descending coronary artery (LAD ligation) and aorta-banding (AB). Sham...

  5. Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion

    DEFF Research Database (Denmark)

    Wree, Dorothea; Wu, Binghua; Zeuthen, Thomas

    2011-01-01

    Two highly conserved NPA motifs are a hallmark of the aquaporin (AQP) family. The NPA triplets form N-terminal helix capping structures with the Asn side chains located in the centre of the water or solute-conducting channel, and are considered to play an important role in AQP selectivity. Although...... another AQP selectivity filter site, the aromatic/Arg (ar/R) constriction, has been well characterized by mutational analysis, experimental data concerning the NPA region--in particular, the Asn position--is missing. Here, we report on the cloning and mutational analysis of a novel aquaglyceroporin...

  6. Characterization of an Aquaporin Gene ZpPIP2 from Zygophyllum potaninii

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    Bayarmaa Gun-Aajav

    2016-11-01

    Full Text Available Zygophyllum potaninii Maxim. is a medicinal plant, distributed in arid regions of southern Mongolia. Full length of a cDNA clone, which was identifi ed as a stress induced gene by suppression subtractive hybridization was obtained by 5’RACEPCR, and named ZpPIP2 as deduced amino acid sequence shows high homology to plant aquaporin PIP2. ZpPIP2 is expressed in leaf and stem under normal conditions, and it is accumulated in the root in response to drought stress in Z. potaninii.

  7. Relationship and action mechanism between oxygen free radicals and aquaporin 4 in brain edema%氧自由基与水通道蛋白4在脑水肿中作用机制及联系

    Institute of Scientific and Technical Information of China (English)

    陈珊; 徐国海

    2011-01-01

    Background Aquaporin-4(AQP4) may be one of the candidates for inducing brain edema,however,it has not been reported whether AQP4 and oxidative free radical is involved in the formation of brain edema.Objective To study the effect as well as the mechanism of oxidative free radical and AQP4 on cerebral edema.Content Aquaporin (AQP) is a membrane water channel protein family.And AQP4 is abundant within the nervous system and is closely related to the physiological and pathological process particularly in the metabolism of water.Perihematoma antioxidant imbalance and oxidative free radical reactions further increase the cerebral edema in acute cerebral hemorrhage.Trend It will provide basis for further exploring of the pathogenesis of cerebral edema by studying the relationship between oxidative free radical and AQP.%背景 水通道蛋白4(aquaporin-4,AQP4)可能是导致脑水肿形成的调节因素之一,但AQP4与氧自由基作用与脑水肿形成尚未见报道.目的 将AQP4与氧自由基在脑水肿的作用及其机制作简要的概述.内容 水通道蛋白(aquaporin,AQP)是膜水通道蛋白家族,其中AQP4在神经系统内含量最丰富,与神经系统生理和病理过程尤其水的代谢密切相关,同时急性脑出血时血肿周围脑组织氧化抗氧化平衡紊乱及自由基反应病理性加剧进一步加重脑水肿.趋向 通过研究氧自由基与APQ关系,进一步为脑水肿的发病机制奠定基础.

  8. Immunopositivity for histone macroH2A1 isoforms marks steatosis-associated hepatocellular carcinoma.

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    Francesca Rappa

    Full Text Available BACKGROUND: Hepatocellular carcinoma (HCC is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously. METHODS: We examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN and the phosphatase and tensin homolog (PTEN liver specific knock-out (KO mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry. RESULTS: Protein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01 in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2, when compared to steatosis (<2% of hepatocytes positive for either isoform. The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2. CONCLUSIONS: These data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.

  9. Isoform specificity of the Na/K-ATPase association and regulation by phospholemman.

    Science.gov (United States)

    Bossuyt, Julie; Despa, Sanda; Han, Fei; Hou, Zhanjia; Robia, Seth L; Lingrel, Jerry B; Bers, Donald M

    2009-09-25

    Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-alpha1 or CFP-NKA-alpha2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 +/- 3.4% for NKA-alpha1 and 27.5 +/- 2.5% for NKA-alpha2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1:1 PLM:NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-alpha2 versus PLM-NKA-alpha1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving >or=3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-alpha1 is ouabain-sensitive and NKA-alpha2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-alpha1 and NKA-alpha2 function. Isoproterenol enhanced internal Na(+) affinity of both isoforms (K((1/2)) decreased from 18.1 +/- 2.0 to 11.5 +/- 1.9 mm for NKA-alpha1 and from 16.4 +/- 2.5 to 10.4 +/- 1.5 mm for NKA-alpha2) without altering maximum transport rate (V(max)). Protein kinase C activation also decreased K((1/2)) for both NKA-alpha1 and NKA-alpha2 (to 9.4 +/- 1.0 and 9.1 +/- 1.1 mm, respectively) but increased V(max) only for NKA-alpha2 (1.9 +/- 0.4 versus 1.2 +/- 0.5 mm/min). In conclusion, PLM associates with and modulates both NKA

  10. Ulipristal Acetate Inhibits Progesterone Receptor Isoform A-Mediated Human Breast Cancer Proliferation and BCl2-L1 Expression.

    Directory of Open Access Journals (Sweden)

    Nathalie Esber

    Full Text Available The progesterone receptor (PR with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4 and ulipristal acetate (UPA, a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL2-L1 and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL2-L1 gene. UPA decreased cell proliferation and repressed BCL2-L1 expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required.

  11. A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain

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    Lalouel Jean-Marc

    2009-04-01

    Full Text Available Abstract Background Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human NEDD4L that generates isoforms that contain or lack a C2 domain. Results To address the potential functional significance of the NEDD4L common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into Xenopus laevis kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence β-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level. Conclusion NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase

  12. Laminin isoforms in endothelial and perivascular basement membranes.

    Science.gov (United States)

    Yousif, Lema F; Di Russo, Jacopo; Sorokin, Lydia

    2013-01-01

    Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis.

  13. Oxygenation properties and isoform diversity of snake hemoglobins

    DEFF Research Database (Denmark)

    Storz, Jay F.; Natarajan, Chandrasekhar; Moriyama, Hideaki

    2015-01-01

    Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer- dimer dissociation. However, standardized comparative data are lacking...... for snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying - and -type globin genes to characterize 1) Hb isoform composition of definitive erythrocytes, and 2......) the oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis...

  14. Proteogenomic Analysis Identifies a Novel Human SHANK3 Isoform

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    Fahad Benthani

    2015-05-01

    Full Text Available Mutations of the SHANK3 gene have been associated with autism spectrum disorder. Individuals harboring different SHANK3 mutations display considerable heterogeneity in their cognitive impairment, likely due to the high SHANK3 transcriptional diversity. In this study, we report a novel interaction between the Mutated in colorectal cancer (MCC protein and a newly identified SHANK3 protein isoform in human colon cancer cells and mouse brain tissue. Hence, our proteogenomic analysis identifies a new human long isoform of the key synaptic protein SHANK3 that was not predicted by the human reference genome. Taken together, our findings describe a potential new role for MCC in neurons, a new human SHANK3 long isoform and, importantly, highlight the use of proteomic data towards the re-annotation of GC-rich genomic regions.

  15. Expression of aquaporin-1 in the human peritoneum and the effect of peritoneal dialysis on its expression

    Institute of Scientific and Technical Information of China (English)

    方炜; 钱家麒; 余志远; 陈诗书

    2003-01-01

    Objective To investigate the expression of aquaporin-1 (AQP1) in the human peritoneum and to evaluate the effect of peritoneal dialysis (PD) on its expression.Methods Peritoneal biopsies were obtained from normal subjects (n=10), uremic nondialysis patients (n=12) at catheter insertion and PD patients (n=10) at the time of catheter removal, reinsertion or renal transplantation. Western blot, immuno-histochemical staining and reverse transcript-polymerase chain reaction (RT-PCR) techniques were used to investigate AQP1 expression.Results All peritoneal samples expressed AQP1 at both mRNA and protein levels. Western blot revealed a major band at 28 kD as well as more diffuse bands between 35 and 50 kD. The 28 kD band represents the nonglycosylated form of the protein while the 35-50 kD bands correspond to glycosylated AQP1. Immunohistochemical staining found the positive deposits were distributed in the mesothelial cells, endothelial cells of capillaries, venules and small veins, whereas no signal was detected in the arterioles. Semi-quantitative analysis showed that AQP1 expression was remarkably stable in all samples, whatever their origin (P>0.05).Conclusions Our findings suggested that AQP1 is the molecular counterpart of an ultra small pore during PD. Secondly, the peritoneal mesothelial cell might also be involved in peritoneal transcellular water transport. As regards whether or not the structural or distributional alterations of AQP1 in the peritoneum may be more obviously expressed during PD, further study is needed.

  16. Expression of VAMP-2-like protein in kidney collecting duct intracellular vesicles. Colocalization with Aquaporin-2 water channels.

    Science.gov (United States)

    Nielsen, S; Marples, D; Birn, H; Mohtashami, M; Dalby, N O; Trimble, M; Knepper, M

    1995-01-01

    Body water balance is controlled by vasopressin, which regulates Aquaporin-2 (AQP2) water channels in kidney collecting duct cells by vesicular trafficking between intracellular vesicles and the plasma membrane. To examine the molecular apparatus involved in vesicle trafficking and vasopressin regulation of AQP2 in collecting duct cells, we tested if targeting proteins expressed in the synaptic vesicles, namely vesicle-associated membrane proteins 1 and 2 (VAMP1 and 2), are expressed in kidney collecting duct. Immunoblotting revealed specific labeling of VAMP2 (18-kD band) but not VAMP1 in membrane fractions prepared from kidney inner medulla. Controls using preadsorbed antibody or preimmune serum were negative. Bands of identical molecular size were detected in immunoblots of brain membrane vesicles and purified synaptic vesicles. VAMP2 in kidney membranes was cleaved by tetanus toxin, revealing a tetanus toxin-sensitive VAMP homologue. Similarly, tetanus toxin cleaved VAMP2 in synaptic vesicles. In kidney inner medulla, VAMP2 was predominantly expressed in the membrane fraction enriched for intracellular vesicles, with little or no VAMP2 in the plasma membrane enriched fraction. This was confirmed by immunocytochemistry using semithin cryosections, which showed mainly vesicular labeling in collecting duct principal cells, with no labeling of intercalated cells. VAMP2 immunolabeling colocalized with AQP2 labeling in intracellular vesicles, as determined by immunoelectron microscopy after double immunolabeling of isolated vesicles. Quantitative analysis of 1,310 vesicles revealed a highly significant association of both AQP2 and VAMP2 in the same vesicles (P < 0.0001). Furthermore, the presence of AQP2 in vesicles immunoisolated with anti-VAMP2 antibodies was confirmed by immunoblotting. In conclusion, VAMP2, a component of the neuronal SNARE complex, is expressed in vesicles carrying AQP2, suggesting a role in vasopressin-regulated vesicle trafficking of AQP2

  17. Morphology and Aquaporin Immunohistochemistry of the Uterine Tube of Saanen Goats (Capra hircus): Comparison Throughout the Reproductive Cycle.

    Science.gov (United States)

    Arrighi, S; Bosi, G; Frattini, S; Coizet, B; Groppetti, D; Pecile, A

    2016-06-01

    The expression of six different aquaporins (AQP1, 2, 3, 4, 5 and 9), integral membrane water channels that facilitate bi-directional passive movement of water, was investigated by immunohistochemistry in the uterine tube of pre-pubertal and adult Saanen goats (Capra hircus), comparing the different phases of the oestrous cycle. Regional morphology and secretory processes were markedly different during the goat oestrous cycle. The tested AQP molecules showed different expression patterns in comparison with already studied species. AQP1-immunoreactivity was evidenced at the endothelium of blood vessels and in nerve fibres, regardless of the tubal tract and cycle period. AQP4-immunoreactivity was shown on the lateral plasmalemma in the basal third of the epithelial cells at infundibulum and ampulla level in the cycling goats, more evidently during follicular than during luteal phase. No AQP4-immunoreactivity was noticed at the level of the isthmus region, regardless of the cycle phase. AQP5-immunoreactivity, localized at the apical surface of epithelial cells, increased from pre-puberty to adulthood. Thereafter, AQP5-immunoreactivity was prominent during the follicular phase, when it strongly decorated the apical plasmalemma of all epithelial cells at ampullary level. During luteal phase, immunoreactivity was discontinuous, being weak to strong at the apex of the secretory cells protruding into the lumen. In the isthmus region, the strongest AQP5-immunoreactivity was seen during follicular phase, with a clear localization in the apical plasmalemma of all the epithelial cells and also on the lateral plasmalemma. AQP2, 3 and 9 were undetectable all along the goat uterine tube. Likely, a collaboration of different AQP molecules sustains the fluid production in the goat uterine tube. AQP1-mediated transudation from the blood capillaries, together with permeation of the epithelium by AQP4 in the basal rim of the epithelial cells and final intervening of apical AQP5, could

  18. Cerebrospinal fluid antibodies to aquaporin-4 in neuromyelitis optica and related disorders: frequency, origin, and diagnostic relevance

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    Jarius Sven

    2010-09-01

    Full Text Available Abstract Background In 70-80% of cases, neuromyelitis optica (NMO is associated with highly specific serum auto-antibodies to aquaporin-4 (termed AQP4-Ab or NMO-IgG. Recent evidence strongly suggests that AQP4-Ab are directly involved in the immunopathogenesis of NMO. Objective To assess the frequency, syndrome specificity, diagnostic relevance, and origin of cerebrospinal fluid (CSF AQP4-Ab in patients with NMO spectrum disorders (NMOSD. Methods 87 CSF samples from 37 patients with NMOSD and 42 controls with other neurological diseases were tested for AQP4-Ab in a cell based assay using recombinant human AQP4. Twenty-three paired CSF and serum samples from AQP4-Ab seropositive NMOSD patients were further analysed for intrathecal IgG synthesis to AQP4. Results AQP4-Ab were detectable in 68% of CSF samples from AQP4-Ab seropositive patients with NMOSD, but in none of the CSF samples from AQP4-Ab seronegative patients with NMOSD and in none of the control samples. Acute disease relapse within 30 days prior to lumbar puncture, AQP4-Ab serum titres >1:250, and blood-CSF barrier dysfunction, but not treatment status, predicted CSF AQP4-Ab positivity. A positive AQP4-specific antibody index was present in 1/23 samples analysed. Conclusions AQP4-Ab are detectable in the CSF of most patients with NMOSD, mainly during relapse, and are highly specific for this condition. In the cohort analysed in this study, testing for CSF AQP4-Ab did not improve the sensitivity and specificity of the current diagnostic criteria for NMO. The substantial lack of intrathecal AQP4-Ab synthesis in patients with NMOSD may reflect the unique localisation of the target antigen at the blood brain barrier, and is important for our understanding of the immunopathogenesis of the disease.

  19. Liver glycerol permeability and aquaporin-9 are dysregulated in a murine model of Non-Alcoholic Fatty Liver Disease.

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    Patrizia Gena

    Full Text Available One form of liver steatosis, namely Non-Alcoholic Fatty Liver Disease (NAFLD, is a worrisome health problem worldwide characterized by intrahepatic triacylglycerol (TG overaccumulation. NAFLD is a common feature of metabolic syndrome being often associated with obesity, dyslipidemia and diabetes and mostly closely linked to insulin resistance. The mechanism of NAFLD pathogenesis is object of intense investigation especially regarding complex systems ultimately resulting in excessive TG deposition in hepatocytes. However, scarce is the attention about the relevance of hepatic import of glycerol, the other primary source (as glycerol-3-phosphate of increased TG in hepatocytes. Obese leptin-deficient (ob/ob mice, an animal model of NAFLD, were used to evaluate the functional involvement of Aquaporin-9 (AQP9, the major pathway of liver glycerol entry, in hepatosteatosis. By RT-PCR and qPCR, the level of Aqp9 mRNA in the liver of starved obese mice was comparable with the corresponding control lean littermates. By immunoblotting, the AQP9 protein at the hepatocyte sinusoidal plasma membrane of obese mice was markedly lower (33% than lean mice, a finding fully confirmed by immunohistochemistry. By stopped-flow light scattering, the liver glycerol permeability of ob/ob mice was significantly lower (53% than lean mice, a finding consistent with both the observed down-regulation of AQP9 protein and increased level of plasma glycerol characterizing obese mice. In summary, our results suggest implication of AQP9 in liver steatosis. The reduction of hepatocyte AQP9 and, consequently, glycerol permeability might be a defensive mechanism to counteract further fat infiltration in liver parenchyma.

  20. Hypoxia-inducible factor-1α contributes to brain edema after stroke by regulating aquaporins and glycerol distribution in brain.

    Science.gov (United States)

    Higashida, Tetsuhiro; Peng, Changya; Li, Jie; Dornbos, David; Teng, Kailing; Li, Xiaohua; Kinni, Harish; Guthikonda, Murali; Ding, Yuchuan

    2011-02-01

    Brain edema following stroke is a critical clinical problem due to its association with increased morbidity and mortality. Despite its significance, present treatment for brain edema simply provides symptomatic relief due to the fact that molecular mechanisms underlying brain edema remain poorly understood. The present study investigated the role of hypoxia-inducible factor-1α (HIF-1α) and aquaporins (AQP-4 and -9) in regulating cerebral glycerol accumulation and inducing brain edema in a rodent model of stroke. Two-hours of middle cerebral artery occlusion (MCAO) followed by reperfusion was performed in male Sprague-Dawley rats (250-280 g). Anti-AQP-4 antibody, anti-AQP-9 antibody, or 2-Methoxyestradiol (2ME2, an inhibitor of HIF-1α) was given at the time of MCAO. The rats were sacrificed at 1 and 24 hours after reperfusion and their brains were examined. Extracellular and intracellular glycerol concentration of brain tissue was calculated with an enzymatic glycerol assay. The protein expressions of HIF-1α, AQP-4 and AQP-9 were determined by Western blotting. Brain edema was measured by brain water content. Compared to control, edema (p < 0.01), increased glycerol (p < 0.05), and enhanced expressions of HIF-1α, AQP-4, and AQP-9 (p < 0.05) were observed after stroke. With inhibition of AQP-4, AQP-9 or HIF-1α, edema and extracellular glycerol were significantly (p < 0.01) decreased while intracellular glycerol was increased (p < 0.01) 1 hour after stroke. Inhibition of HIF-1α with 2ME2 suppressed (p < 0.01) the expression of AQP-4 and AQP-9. These findings suggest that HIF-1α serves as an upstream regulator of cerebral glycerol concentrations and brain edema via a molecular pathway involving AQP-4 and AQP-9. Pharmacological blockade of this pathway in stroke patients may provide novel therapeutic strategies.

  1. Functional characterization of two secreted SEL1L isoforms capable of exporting unassembled substrate.

    Science.gov (United States)

    Cattaneo, Monica; Lotti, Lavinia Vittoria; Martino, Simone; Cardano, Marina; Orlandi, Rosaria; Mariani-Costantini, Renato; Biunno, Ida

    2009-04-24

    SEL1L-A, a transmembrane glycoprotein residing in the endoplasmic reticulum (ER), is a component of the ER-associated degradation (ERAD) pathway. Alternative splicing generates two smaller SEL1L isoforms, -B and -C, that lack the SEL1L-A membrane-spanning region but retain some sel-1-like repeats, known to be involved in multi-protein interactions and signal transduction. In this study the functional characteristics of SEL1L-B and -C were investigated in human cell models. We show that these two isoforms are induced upon ER stress and activation of the unfolded protein response, together with SEL1L-A. Using transient transfection experiments (based on wild-type and mutant SEL1L constructs) combined with several biochemical tests we show that SEL1L-B and, more prominently, SEL1L-C are secreted glycoproteins. Although SEL1L-C is in monomeric form, SEL1L-B is engaged in intramolecular/intermolecular disulfide bonds. Both isoforms localize in secretory and degradative cellular compartments and in areas of cell-cell contact. However, whereas SEL1L-B is mainly associated with membranes, SEL1L-C shows the typical intralumenal localization of soluble proteins and is present in intercellular spaces. Furthermore, because of its peroxisomal domain, SEL1L-C localizes to peroxisomes. Both SEL1L-B and -C are involved in sorting and exporting unassembled Ig-mu(s) but do not affect two other ERAD substrates, the null Hong Kong variant of alpha(1)-antitrypsin, and mutant alpha(1)-AT Z. Overall these findings suggest that SEL1L-B and -C participate to novel molecular pathways that, in parallel with ERAD, contribute to the disposure of misfolded/unfolded or orphan proteins through degradation or secretion.

  2. Functional Characterization of Two Secreted SEL1L Isoforms Capable of Exporting Unassembled Substrate*S⃞

    Science.gov (United States)

    Cattaneo, Monica; Lotti, Lavinia Vittoria; Martino, Simone; Cardano, Marina; Orlandi, Rosaria; Mariani-Costantini, Renato; Biunno, Ida

    2009-01-01

    SEL1L-A, a transmembrane glycoprotein residing in the endoplasmic reticulum (ER), is a component of the ER-associated degradation (ERAD) pathway. Alternative splicing generates two smaller SEL1L isoforms, -B and -C, that lack the SEL1L-A membrane-spanning region but retain some sel-1-like repeats, known to be involved in multi-protein interactions and signal transduction. In this study the functional characteristics of SEL1L-B and -C were investigated in human cell models. We show that these two isoforms are induced upon ER stress and activation of the unfolded protein response, together with SEL1L-A. Using transient transfection experiments (based on wild-type and mutant SEL1L constructs) combined with several biochemical tests we show that SEL1L-B and, more prominently, SEL1L-C are secreted glycoproteins. Although SEL1L-C is in monomeric form, SEL1L-B is engaged in intramolecular/intermolecular disulfide bonds. Both isoforms localize in secretory and degradative cellular compartments and in areas of cell-cell contact. However, whereas SEL1L-B is mainly associated with membranes, SEL1L-C shows the typical intralumenal localization of soluble proteins and is present in intercellular spaces. Furthermore, because of its peroxisomal domain, SEL1L-C localizes to peroxisomes. Both SEL1L-B and -C are involved in sorting and exporting unassembled Ig-μs but do not affect two other ERAD substrates, the null Hong Kong variant of α1-antitrypsin, and mutant α1-AT Z. Overall these findings suggest that SEL1L-B and -C participate to novel molecular pathways that, in parallel with ERAD, contribute to the disposure of misfolded/unfolded or orphan proteins through degradation or secretion. PMID:19204006

  3. CEACAM1 Long Cytoplasmic Domain Isoform is Associated with Invasion and Recurrence of Hepatocellular Carcinoma

    Science.gov (United States)

    Kiriyama, Shigehisa; Yokoyama, Shozo; Ueno, Masaki; Hayami, Shinya; Ieda, Junji; Yamamoto, Naoyuki; Yamaguchi, Shunsuke; Mitani, Yasuyuki; Nakamura, Yasushi; Tani, Masaji; Mishra, Lopa; Shively, John E.; Yamaue, Hiroki

    2014-01-01

    Background The two isoforms of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), 1 with a long cytoplasmic domain (CEACAM1-L) and 1 with a short (CEACAM1-S), are involved in different signaling pathways. β2-spectrin (β2SP) is an adaptor protein that plays critical roles in the proper control of Smad access to activate receptors involved in regulation of TGF-β signaling. In this study, we examined the association between CEACAM1 isoform balance and hepatocellular carcinoma (HCC) malignant potential and investigated the possibility of a molecular interaction between CEACAM1 and β2SP. Methods Immunohistochemical analysis was carried out with CEACAM1-L or CEACAM1-S antibodies on 154 HCC tissues to correlate with the factors of malignancy. Invasion assay was performed for the effect of CEACAM1 expression on HCC cell lines. Moreover, immunohistochemical analysis and immunoprecipitation analysis were performed to investigate the association between CEACAM1 isoform balance and β2SP. Results In immunohistochemical analysis, CEACAM1-L expression dominance was a risk factor for HCC recurrence (p = 0.04) and was significantly associated with a shorter survival compared with CEACAM1-S expression dominance. Invasion assay indicated that CEACAM1-4L-transfected HLF and PLC/PRF/5 cells showed significantly increased invasion (p<0.0001) and CEACAM1-4S-transfected HLF cells showed significantly decreased invasion. Immunohistochemical analysis of β2SP suggested that the HCCs with CEACAM1-L-dominant expression were more strongly stained with β2SP than the HCCs with CEACAM1-S-dominant expression (p = 0.013), and coprecipitation assays indicated that CEACAM1-L could bind to β2SP. Conclusions CEACAM1-L may enhance the HCC invasiveness through an interaction with β2SP and subsequent effects on TGF-β signaling. PMID:24390710

  4. Characterization of IGF-II isoforms in binge eating disorder and its group psychological treatment.

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    Giorgio Tasca

    Full Text Available Binge eating disorder (BED affects 3.5% of the population and is characterized by binge eating for at least 2 days a week for 6 months. Treatment options include cognitive behavioral therapy, interpersonal psychotherapy, and pharmacotherapy which are associated with varied success. Little is known about the biology of BED. Since there is evidence that the insulin like growth factor system is implicated in regulation of body weight, insulin sensitivity and feeding behavior, we speculated it may be involved in BED.A cross-sectional comparison was made between three groups of women: overweight with BED, overweight without BED and normal weight without BED. Women were assigned to Group Psychodynamic Interpersonal Psychotherapy. Blood was collected before therapy, at completion and at 6 months follow up for evaluation of IGF-II using Western blot.97 overweight women with BED contributed to the cross-sectional comparison. The two control groups comprised 53 overweight women without BED, and 50 age matched normal weight women without BED. Obese women had significantly lower Big IGF-II than normal weight women, p = .028; Overweight women with BED had higher Mature IGF-II than normal weight women, p<.05. Big IGF-II showed a significant decreasing slope from pre- to post- to six months post-group psychological treatment, unrelated to changes in BMI (p = .008.Levels of IGF-II isoforms differed significantly between overweight and normal weight women. Overweight women with BED display abnormal levels of circulating IGF-II isoforms. BED is characterized by elevated mature IGF-II, an isoform shown to carry significant bioactivity. This finding is not related to BMI or to changes in body weight. The results also provide preliminary evidence that BIG IGF-II is sensitive to change due to group psychological treatment. We suggest that abnormalities in IGF-II processing may be involved in the neurobiology of BED.

  5. Identification and characterization of novel NuMA isoforms

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jin, E-mail: petersdu2112@hotmail.com [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Xu, Zhe [Department of Clinical Laboratory Diagnosis, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); Core Laboratory for Clinical Medical Research, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); He, Dacheng [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Lu, Guanting, E-mail: guantlv@126.com [Beijing DnaLead Science and Technology Co., LTD, Beijing (China)

    2014-11-21

    Highlights: • Seven NuMA isoforms generated by alternative splicing were categorized into 3 groups: long, middle and short. • Both exons 15 and 16 in long NuMA were “hotspot” for alternative splicing. • Lower expression of short NuMA was observed in cancer cells compared with nonneoplastic controls. • Distinct localization pattern of short isoforms indicated different function from that of long and middle NuMA. - Abstract: The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.

  6. A novel mutation affecting the arginine-137 residue of AVPR2 in dizygous twins leads to nephrogenic diabetes insipidus and attenuated urine exosome aquaporin-2

    DEFF Research Database (Denmark)

    Hinrichs, Gitte R; Hansen, Louise H; Nielsen, Maria R

    2016-01-01

    Mutations in the vasopressin V2 receptor gene AVPR2 may cause X-linked nephrogenic diabetes insipidus by defective apical insertion of aquaporin-2 in the renal collecting duct principal cell. Substitution mutations with exchange of arginine at codon 137 can cause nephrogenic syndrome of inappropr...... administration. While a similar urine exosome release rate was shown between probands and controls by western blotting for the marker ALIX, there was a selective decrease in exosome aquaporin-2 versus aquaporin-1 protein in probands compared to controls....

  7. Aquaporins 7 and 11 in boar spermatozoa: detection, localisation and relationship with sperm quality.

    Science.gov (United States)

    Prieto-Martínez, Noelia; Vilagran, Ingrid; Morató, Roser; Rodríguez-Gil, Joan E; Yeste, Marc; Bonet, Sergi

    2016-04-01

    Aquaporins (AQPs) are integral membrane water channels that allow transport of water and small solutes across cell membranes. Although water permeability is known to play a critical role in mammalian cells, including spermatozoa, little is known about their localisation in boar spermatozoa. Two aquaporins, AQP7 and AQP11, in boar spermatozoa were identified by western blotting and localised through immunocytochemistry analyses. Western blot results showed that boar spermatozoa expressed AQP7 (25kDa) and AQP11 (50kDa). Immunocytochemistry analyses demonstrated that AQP7 was localised in the connecting piece of boar spermatozoa, while AQP11 was found in the head and mid-piece and diffuse labelling was also seen along the tail. Despite differences in AQP7 and AQP11 content between boar ejaculates, these differences were not found to be correlated with sperm quality in the case of AQP7. Conversely, AQP11 content showed a significant correlation (Psperm membrane integrity and fluidity and sperm motility. In conclusion, boar spermatozoa express AQP7 and AQP11, and the amounts of AQP11 but not those of AQP7 are correlated with sperm motility and membrane integrity.

  8. Importance of NPA motifs in the expression and function of water channel aquaporin-1

    Institute of Scientific and Technical Information of China (English)

    JIANG Yong; MA TongHui

    2007-01-01

    The asparagine-proline-alanine sequences (NPA motifs) are highly conserved in aquaporin water channel family. Crystallographic studies of AQP1 structure demonstrated that the two NPA motifs are in the narrow central constriction of the channel, serving to bind water molecules for selective and efficient water passage. To investigate the importance of the two NPA motifs in the structure, function and biogenesis of aquaporin water channels, we generated AQP1 mutations with NPA1 deletion, NPA2 deletion and NPA1,2 double deletion. The coding sequences of the three mutated cDNAs were subcloned into the mammalian expression vector pcDNA3.1 to form expression plasmids. We established stably transfected CHO cell lines expressing these AQP1 mutants. Immunofluorescence indicated that all the three mutated AQP1 proteins are expressed normally on the plasma membrane of stably transfected CHO cells, suggesting that deletion of NPA motifs does not influence the expression and intracellular processing of AQP1. Functional analysis demonstrated that NPA1 or NPA2 deletion reduced AQP1 water permeability by 49.6% and 46.7%, respectively, while NPA1,2 double deletion had little effect on AQP1 water permeability. These results provide evidence that NPA motifs are important for water per-meation but not essential for the expression, intracellular processing and the basic structure of AQP1 water channel.

  9. Toward understanding the selective anticancer capacity of cold atmospheric plasma--a model based on aquaporins (Review).

    Science.gov (United States)

    Yan, Dayun; Talbot, Annie; Nourmohammadi, Niki; Sherman, Jonathan H; Cheng, Xiaoqian; Keidar, Michael

    2015-01-01

    Selectively treating tumor cells is the ongoing challenge of modern cancer therapy. Recently, cold atmospheric plasma (CAP), a near room-temperature ionized gas, has been demonstrated to exhibit selective anticancer behavior. However, the mechanism governing such selectivity is still largely unknown. In this review, the authors first summarize the progress that has been made applying CAP as a selective tool for cancer treatment. Then, the key role of aquaporins in the H2O2 transmembrane diffusion is discussed. Finally, a novel model, based on the expression of aquaporins, is proposed to explain why cancer cells respond to CAP treatment with a greater rise in reactive oxygen species than homologous normal cells. Cancer cells tend to express more aquaporins on their cytoplasmic membranes, which may cause the H2O2 uptake speed in cancer cells to be faster than in normal cells. As a result, CAP treatment kills cancer cells more easily than normal cells. Our preliminary observations indicated that glioblastoma cells consumed H2O2 much faster than did astrocytes in either the CAP-treated or H2O2-rich media, which supported the selective model based on aquaporins.

  10. Aquaporin 2 of Rhipicephalus (Boophilus) microplus as a potential target to control ticks and tick-borne parasites

    Science.gov (United States)

    In a collaboration with Washington State University and ARS-Pullman, WA researchers, we identified and sequenced a 1,059 base pair Rhipicephalus microplus transcript that contained the coding region for a water channel protein, Aquaporin 2 (RmAQP2). The clone sequencing resulted in the production of...

  11. Immunogenic potential of the recombinant Rhipicephalus microplus aquaporin protein against the tick Rhipicephalus sanguineus Latreille, 1806 in domestic dogs

    Science.gov (United States)

    Aquaporins regulate water transport through the highly hydrophobic lipid bilayer of cell membranes. As ticks ingest large volumes of host blood in relation to their size, they are required to concentrate blood components and have efficient water transport mechanisms. This study aimed to evaluate the...

  12. Influence of low air humidity and low root temperature on water uptake, growth and aquaporin expression in rice plants.

    Science.gov (United States)

    Kuwagata, Tsuneo; Ishikawa-Sakurai, Junko; Hayashi, Hidehiro; Nagasuga, Kiyoshi; Fukushi, Keiko; Ahamed, Arifa; Takasugi, Katsuko; Katsuhara, Maki; Murai-Hatano, Mari

    2012-08-01

    The effects of low air humidity and low root temperature (LRT) on water uptake, growth and aquaporin gene expression were investigated in rice plants. The daily transpiration of the plants grown at low humidity was 1.5- to 2-fold higher than that at high humidity. LRT at 13°C reduced transpiration, and the extent was larger at lower humidity. LRT also reduced total dry matter production and leaf area expansion, and the extent was again larger at lower humidity. These observations suggest that the suppression of plant growth by LRT is associated with water stress due to decreased water uptake ability of the root. On the other hand, the net assimilation rate was not affected by low humidity and LRT, and water use efficiency was larger for LRT. We found that low humidity induced coordinated up-regulation of many PIP and TIP aquaporin genes in both the leaves and the roots. Expression levels of two root-specific aquaporin genes, OsPIP2;4 and OsPIP2;5, were increased significantly after 6 and 13 d of LRT exposure. Taken together, we discuss the possibility that aquaporins are part of an integrated response of this crop to low air humidity and LRT.

  13. The role of putative phosphorylation sites in the targeting and shuttling of the aquaporin-2 water channel.

    NARCIS (Netherlands)

    Balkom, B.W.M. van; Savelkoul, P.J.M.; Markovich, D.; Hofman, E.; Nielsen, S.; Sluijs, P. van der; Deen, P.M.T.

    2002-01-01

    In renal collecting ducts, a vasopressin-induced cAMP increase results in the phosphorylation of aquaporin-2 (AQP2) water channels at Ser-256 and its redistribution from intracellular vesicles to the apical membrane. Hormones that activate protein kinase C (PKC) proteins counteract this process. To

  14. Correlation of aquaporin-4 expression to blood-brain barrier permeability in rats with focal cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Pengcheng Xu; Haorong Feng; Jinbu Xu; Yongping Wu

    2008-01-01

    BACKGROUND: Ischemic cerebrovascular disease causes injury to the blood-brain barrier. The occurrence of brain edema is associated with aquaporin expression following cerebral ischemia/reperfusion. OBJECTIVE: To analyze the correlation of aquaporin-4 expression to brain edema and blood-brain barrier permeability in brain tissues of rat models of ischemia/reperfusion. DESIGN, TIME AND SETTING: The randomized control experiment was performed at the Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, China from December 2006 to October 2007. MATERIALS: A total of 112 adult, male, Sprague-Dawley rats, weighing 220-250 g, were used to establish rat models of middle cerebral artery occlusion and reperfusion by the suture method. Rabbit anti-aquaporin-4 (Santa Cruz, USA) and Evans blue (Sigma, USA) were used to analyze the tissue. METHODS: The rats were randomized into sham-operated (n = 16) and ischemia/reperfusion (n = 96) groups. There were 6 time points in the ischemia/reperfusion group, comprising 4, 6, 12, 24, 48, and 72 hours after reperfusion, with 16 rats for each time point. Rat models in the sham-operated group at 4 hours after surgery and rat models in the ischemia/reperfusion group at different time points were equally and randomly assigned into 4 different subgroups. MAIN OUTCOME MEASURES: Brain water content on the ischemic side and the control side was measured using the dry-wet weight method. Blood-brain barrier function was determined by Evans Blue. Aquaporin-4 expression surrounding the ischemic focus, as well as the correlation of aquaporin-4 expression with brain water content and Evans blue staining, were measured using immunohistochemistry and Western blot analysis. RESULTS: Brain water content on the ischemic side significantly increased at 12 hours after reperfusion, reached a peak at 48 hours, and was still high at 72 hours. Brain water content was greater on the ischemic hemispheres, compared with the control hemispheres

  15. Managing brain extracellular K+ during neuronal activity: The physiological role of the Na+/K+-ATPase subunit isoforms

    Directory of Open Access Journals (Sweden)

    Brian Roland eLarsen

    2016-04-01

    Full Text Available AbstractDuring neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity.Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2 and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood. This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease.

  16. Runx2-I isoform contributes to fetal bone formation even in the absence of specific N-terminal amino acids.

    Directory of Open Access Journals (Sweden)

    Hideaki Okura

    Full Text Available The Runt-related transcription factor 2 (Runx2 gene encodes the transcription factor Runx2, which is the master regulator of osteoblast development; insufficiency of this protein causes disorders of bone development such as cleidocranial dysplasia. Runx2 has two isoforms, Runx2-II and Runx2-I, and production of each isoform is controlled by a unique promoter: a distal promoter (P1 and a proximal promoter (P2, respectively. Although several studies have focused on differences and similarities between the two Runx2 isoforms, their individual roles in bone formation have not yet been determined conclusively, partly because a Runx2-I-targeted mouse model is not available. In this study, we established a novel Runx2-manipulated mouse model in which the first ATG of Runx2-I was replaced with TGA (a stop codon, and a neomycin-resistant gene (neo cassette was inserted at the first intron of Runx2-I. Homozygous Runx2-Ineo/neo mice showed severely reduced expression of Runx2-I, whereas Runx2-II expression was largely retained. Runx2-Ineo/neo mice showed neonatal lethality, and in these mice, intramembranous ossification was more severely defective than endochondral ossification, presumably because of the greater involvement of Runx2-I, compared with that of Runx2-II in intramembranous ossification. Interestingly, the depletion of neo rescued the above-described phenotypes, indicating that the isoform-specific N-terminal region of Runx2-I is not functionally essential for bone development. Taken together, our results provide a novel clue leading to a better understanding of the roles of Runx2 isoforms in osteoblast development.

  17. Systematically differentiating functions for alternatively spliced isoforms through integrating RNA-seq data.

    Science.gov (United States)

    Eksi, Ridvan; Li, Hong-Dong; Menon, Rajasree; Wen, Yuchen; Omenn, Gilbert S; Kretzler, Matthias; Guan, Yuanfang

    2013-01-01

    Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires 'ground-truth' functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the 'responsible' isoform(s) of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the 'responsible' isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions.

  18. Systematically differentiating functions for alternatively spliced isoforms through integrating RNA-seq data.

    Directory of Open Access Journals (Sweden)

    Ridvan Eksi

    Full Text Available Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires 'ground-truth' functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the 'responsible' isoform(s of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the 'responsible' isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions.

  19. Grapevine aquaporins: gating of a tonoplast intrinsic protein (TIP2;1 by cytosolic pH.

    Directory of Open Access Journals (Sweden)

    Luís Leitão

    Full Text Available Grapevine (Vitis vinifera L. is one of the oldest and most important perennial crops being considered as a fruit ligneous tree model system in which the water status appears crucial for high fruit and wine quality, controlling productivity and alcohol level. V. vinifera genome contains 28 genes coding for aquaporins, which acting in a concerted and regulated manner appear relevant for plant withstanding extremely unfavorable drought conditions essential for the quality of berries and wine. Several Vv aquaporins have been reported to be expressed in roots, shoots, berries and leaves with clear cultivar differences in their expression level, making their in vivo biochemical characterization a difficult task. In this work V. vinifera cv. Touriga nacional VvTnPIP1;1, VvTnPIP2;2 and VvTnTIP2;1 were expressed in yeast and water transport activity was characterized in intact cells of the transformants. The three aquaporins were localized in the yeast plasma membrane but only VvTnTIP2;1 expression enhanced the water permeability with a concomitant decrease of the activation energy of water transport. Acidification of yeast cytosol resulted in loss of VvTnTIP2;1 activity. Sequence analysis revealed the presence of a His(131 residue, unusual in TIPs. By site directed mutagenesis, replacement of this residue by aspartic acid or alanine resulted in loss of pH(in dependence while replacement by lysine resulted in total loss of activity. In addition to characterization of VvTn aquaporins, these results shed light on the gating of a specific tonoplast aquaporin by cytosolic pH.

  20. Isoforms of transferrin in psoriasis patients abusing alcohol

    NARCIS (Netherlands)

    P. Hoefkens (Peter); E.M. Higgins; R.J. Ward (Roberta); H.G. van Eijk (Henk)

    1997-01-01

    textabstractThe different isoforms of transferrin have been quantified by isoelectric focusing in the sera of psoriasis patients with and without a history of abusing alcohol. In both male and female psoriasis subjects abusing alcohol, there were significant increases in the 2-sial

  1. Murine Sirt3 protein isoforms have variable half-lives

    Science.gov (United States)

    Sirt3 is a NAD+-dependent protein deacetylase mainly localized in mitochondria. Recent studies indicate that the murine Sirt3 gene expresses different transcript variants resulting in three possible Sirt3 protein isoforms with variable lengths at the N-terminus: M1 (aa 1-334), M2 (aa 15-334), and M3...

  2. Protein tyrosine phosphatase PTPRR isoforms in cellular signaling and trafficking

    NARCIS (Netherlands)

    Dilaver, Gönül

    2005-01-01

    Previous work has revealed the existence of two Protein Tyrosine Phosphatases in mouse, PTPBR7 and PTP-SL, that were in part identical, suggesting that they originated from the same gene, termed Ptprr (1,5,6). In this thesis, I report on the characterization of the various PTPRR isoforms in neuronal

  3. Identification and characterization of a novel isoform of hepatopoietin

    Institute of Scientific and Technical Information of China (English)

    Jun Lu; Wang-Xiang Xu; Yi-Qun Zhan; Xiao-Lin Cui; Wei-Min Cai; Fu-Chu He; Xiao-Ming Yang

    2002-01-01

    AIM: To isolate a novel isoform of human HPO (HPO-205)human fetal liver Marathon-reedy cDNA andcharacterize its primary biological function.METHODS: 5'-RACE (rapid amplification of cDNA 5' ends)was used to isolate a novel isoform of hHPO in this paperThe constructed pcDNAHPO-205, pcDNAHPO and pcDNA eukaryotic expression vectors were respectively transfectedby lipofectamine method and the stimulation of DNAsynthesis was observed by 3H-TdR incorporation assay.Proteins extracted from different cells were analyzed byWestern blot.RESULTS: A novel isoform of hHPO (HPO-205) encoding a205 amino acid ORF corresponding to a translatedproduction of 23 kDa was isolated and distinguished fromthe previous HPO that lacked the N-terminal 80 amino acids.The dnse-dspendent stimulation of DNA synthesis of HepG2hepatoma cells by HPO-205 demonstrated its similarbiological activity with HPO in vitro. The level of MAPK(Mitogen-activated protein kinase) phnsphorylarion byWestern blot analysis revealed that HPO-205 might have thestronger activity of stimulating hepatic cell proliferation thanthat of HPO.CONCLUSION: A novel isoform of hHPO (HPO-205) wasisolated from hepatic-derived cells. The comparison of HPO-205 and HPO will lead to a new insight into the structure andfunction of hHPO, and provide the new way of thinking todeeply elucidate the biological roles of HPO/ALR.

  4. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

    Directory of Open Access Journals (Sweden)

    Jifeng Zhang

    2015-01-01

    Full Text Available Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca2+ channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons.

  5. Overexpression of rice glutaredoxins (OsGrxs) significantly reduces arsenite accumulation by maintaining glutathione pool and modulating aquaporins in yeast.

    Science.gov (United States)

    Verma, Pankaj Kumar; Verma, Shikha; Meher, Alok Kumar; Pande, Veena; Mallick, Shekhar; Bansiwal, Amit Kumar; Tripathi, Rudra Deo; Dhankher, Om Parkash; Chakrabarty, Debasis

    2016-09-01

    Arsenic (As) is an acute poison and class I carcinogen, can cause a serious health risk. Staple crops like rice are the primary source of As contamination in human food. Rice grown on As contaminated areas accumulates higher As in their edible parts. Based on our previous transcriptome data, two rice glutaredoxins (OsGrx_C7 and OsGrx_C2.1) were identified that showed up-regulated expression during As stress. Here, we report OsGrx_C7 and OsGrx_C2.1 from rice involved in the regulation of intracellular arsenite (AsIII). To elucidate the mechanism of OsGrx mediated As tolerance, both OsGrxs were cloned and expressed in Escherichia coli (Δars) and Saccharomyces cerevisiae mutant strains (Δycf1, Δacr3). The expression of OsGrxs increased As tolerance in E. coli (Δars) mutant strain (up to 4 mM AsV and up to 0.6 mM AsIII). During AsIII exposure, S. cerevisiae (Δacr3) harboring OsGrx_C7 and OsGrx_C2.1 have lower intracellular AsIII accumulation (up to 30.43% and 24.90%, respectively), compared to vector control. Arsenic accumulation in As-sensitive S. cerevisiae mutant (Δycf1) also reduced significantly on exposure to inorganic As. The expression of OsGrxs in yeast maintained intracellular GSH pool and increased extracellular GSH concentration. Purified OsGrxs displays in vitro GSH-disulfide oxidoreductase, glutathione reductase and arsenate reductase activities. Also, both OsGrxs are involved in AsIII extrusion by altering the Fps1 transcripts in yeast and protect the cell by maintaining cellular GSH pool. Thus, our results strongly suggest that OsGrxs play a crucial role in the maintenance of the intracellular GSH pool and redox status of the cell during both AsV and AsIII stress and might be involved in regulating intracellular AsIII levels by modulation of aquaporin expression and functions.

  6. p53 and its isoforms in cancer

    OpenAIRE

    2007-01-01

    p53, p63 and p73 are members of the p53 gene family involved in development, differentiation and response to cellular stress. p53 gene is a transcription factor essential for the prevention of cancer formation. The p53 pathway is ubiquitously lost in human cancer either by p53 gene mutation (60% of cancers) or by lost of cell signalling upstream and downstream of p53 in the remaining cancers expressing WTp53 gene. As p53 pathway inactivation is a common denominator to all cancers, the underst...

  7. The crystal structure of the non-liganded 14-3-3σ protein: insights into determinants of isoform specific ligand binding and dimerization

    Institute of Scientific and Technical Information of China (English)

    Anne BENZINGER; Grzegorz M. POPOWICZ; Joma K. JOY; Sudipta MAJUMDAR; Tad A. HOLAK; Heiko HERMEKING

    2005-01-01

    Seven different, but highly conserved 14-3-3 proteins are involved in diverse signaling pathways in human cells. It is unclear how the 14-3-3σ isoform, a transcriptional target of p53, exerts its inhibitory effect on the cell cycle in the presence of other 14-3-3 isoforms, which are constitutively expressed at high levels. In order to identify structural differences between the 14-3-3 isoforms, we solved the crystal structure of the human 14-3-3σ protein at a resolution of 2.8 A and compared it to the known structures of 14-3-3ζ and 14-3-3τ. The global architecture of the 14-3-3σ fold is similar to the previously determined structures of 14-3-3ζ and 14-3-3τ: two 14-3-3σ molecules form a cup-shaped dimer. Significant differences between these 14-3-3 isoforms were detected adjacent to the amphipathic groove, which mediates the binding to phosphorylated consensus motifs in 14-3-3-1igands. Another specificity determining region is localized between amino-acids 203 to 215. These differences presumably select for the interaction with specific ligands,which may explain the different biological functions of the respective 14-3-3 isoforms. Furthermore, the two 14-3-3σ molecules forming a dimer differ by the spatial position of the ninth helix, which is shifted to the inside of the ligand interaction surface, thus indicating adaptability of this part of the molecule. In addition, 5 non-conserved residues are located at the interface between two 14-3-3σ proteins forming a dimer and represent candidate determinants of homoand hetero-dimerization specificity. The structural differences among the 14-3-3 isoforms described here presumably contribute to isoform-specific interactions and functions.

  8. Cloning, expression and alternative splicing of the novel isoform of hTCP11 gene

    DEFF Research Database (Denmark)

    Ma, Yong-xin; Zhang, Si-zhong; Wu, Qia-qing;

    2003-01-01

    To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing.......To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing....

  9. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons

    Energy Technology Data Exchange (ETDEWEB)

    Fujimoto, Takahiro; Itoh, Kyoko, E-mail: kxi14@koto.kpu-m.ac.jp; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    Highlights: • Identification of dystrophin (Dp) shortest isoform, Dp40, is a neuron-type Dp. • Dp40 expression is temporally and differentially regulated in comparison to Dp71. • Somatodendritic and nuclear localization of Dp40. • Dp40 is localized to excitatory postsynapses. • Dp40 might play roles in dendritic and synaptic functions. - Abstract: The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH{sub 2}-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

  10. Differential induction of FosB isoforms throughout the brain by fluoxetine and chronic stress.

    Science.gov (United States)

    Vialou, Vincent; Thibault, Mackenzie; Kaska, Sophia; Cooper, Sarah; Gajewski, Paula; Eagle, Andrew; Mazei-Robison, Michelle; Nestler, Eric J; Robison, A J

    2015-12-01

    Major depressive disorder is thought to arise in part from dysfunction of the brain's "reward circuitry", consisting of the mesolimbic dopamine system and the glutamatergic and neuromodulatory inputs onto this system. Both chronic stress and antidepressant treatment regulate gene transcription in many of the brain regions that make up these circuits, but the exact nature of the transcription factors and target genes involved in these processes remain unclear. Here, we demonstrate induction of the FosB family of transcription factors in ∼25 distinct regions of adult mouse brain, including many parts of the reward circuitry, by chronic exposure to the antidepressant fluoxetine. We further uncover specific patterns of FosB gene product expression (i.e., differential expression of full-length FosB, ΔFosB, and Δ2ΔFosB) in brain regions associated with depression--the nucleus accumbens (NAc), prefrontal cortex (PFC), and hippocampus--in response to chronic fluoxetine treatment, and contrast these patterns with differential induction of FosB isoforms in the chronic social defeat stress model of depression with and without fluoxetine treatment. We find that chronic fluoxetine, in contrast to stress, causes induction of the unstable full-length FosB isoform in the NAc, PFC, and hippocampus even 24 h following the final injection, indicating that these brain regions may undergo chronic activation when fluoxetine is on board, even in the absence of stress. We also find that only the stable ΔFosB isoform correlates with behavioral responses to stress. These data suggest that NAc, PFC, and hippocampus may present useful targets for directed intervention in mood disorders (ie, brain stimulation or gene therapy), and that determining the gene targets of FosB-mediated transcription in these brain regions in response to fluoxetine may yield novel inroads for pharmaceutical intervention in depressive disorders.

  11. Sequential activation of protein kinase C isoforms by organic dust is mediated by tumor necrosis factor.

    Science.gov (United States)

    Wyatt, Todd A; Slager, Rebecca E; Heires, Arthur J; Devasure, Jane M; Vonessen, Susanna G; Poole, Jill A; Romberger, Debra J

    2010-06-01

    Dust samples collected from Nebraska swine confinement facilities (hog dust extract [HDE]) are known to elicit proinflammatory cytokine release from human bronchial epithelial (HBE) cells in vitro. This response involves the activation of two protein kinase C (PKC) isoforms: PKCalpha and PKCepsilon. Experiments were designed to investigate the relationship between the two isoenzymes and the degree to which each is responsible for cytokine release in HBE. Experiments also examined the contribution of TNF-alpha to IL-6 and IL-8 release. PKCalpha and PKCepsilon activities were inhibited using isoform-specific pharmacologic inhibitors and genetically modified dominant-negative (DN) expressing cell lines. Release of the proinflammatory cytokines IL-6, IL-8, and TNF-alpha was measured and PKC isoform activities assessed. We found that HDE stimulates PKCalpha activity by 1 hour, and within 6 hours the activity returns to baseline. PKCalpha-specific inhibitor or PKCalphaDN cells abolish this HDE-mediated effect. Both IL-6 and IL-8 release are likewise diminished under these conditions compared with normal HBE, and treatment with TNF-alpha-neutralizing antibody does not further inhibit cytokine release. In contrast, PKCepsilon activity was enhanced by 6 hours after HDE treatment. TNF-alpha blockade abrogated this effect. HDE-stimulated IL-6, but not IL-8 release in PKCepsilonDN cells. The concentration of TNF-alpha released by HDE-stimulated HBE is sufficient to have a potent cytokine-eliciting effect. A time course of TNF-alpha release suggests that TNF-alpha is produced after PKCalpha activation, but before PKCepsilon. These results suggest a temporal ordering of events responsible for the release of cytokines, which initiate and exacerbate inflammatory events in the airways of people exposed to agricultural dust.

  12. The Frequency of Anti-Aquaporin-4 Ig G Antibody in Neuromyelitis Optica and Its Spectrum Disorders at a Single Tertiary Referral Center in Malaysia

    Directory of Open Access Journals (Sweden)

    Shanthi Viswanathan

    2014-01-01

    Full Text Available Background. In the past the occurrence of neuromyelitis optica in Malaysia was thought to be uncommon and the frequency of anti-aquaporin-4 Ig G antibody was unknown. Objective. To evaluate the frequency of anti-aquaporin-4 Ig G antibody (Anti-AQP4 antibody amongst patients with neuromyelitis optica (NMO and its spectrum disorders (NMOSD and the differences between the seropositive and seronegative groups. Methods. Retrospectively, 96 patients with NMO/high risk syndromes for NMOSD (HRS-NMOSD were identified out of 266 patients with idiopathic inflammatory demyelinating disease from a single center hospital based registry. Anti-AQP4 seropositivity was found in 38/48 (79.2% with NMO, 12/21 (57.1% with brain involvement at high risk for NMOSD, 12/15 (80% with transverse myelitis (i.e., 11/15 with relapsing transverse myelitis and one with monophasic transverse myelitis, and 3/7 (42.8% with relapsing optic neuritis. Sixty-five out of 96 patients, that is, 67.7%, with NMO/HRS for NMOSD were seropositive. Seropositivity was significantly associated with female gender, a higher number of mean relapses, that is, 5.15 ± 4.42 versus 2.10 ± 1.68, longer length of spinal cord lesions, that is, 6.6 ± 4.9 versus 2.9 ± 2.5, vertebral bodies, higher EDSS, 4.5 ± 2.4 versus 2.4 ± 2.6, presence of paroxysmal tonic spasms, and blindness (unilateral/bilateral; P<0.001. Longitudinally extensive cord lesions (contiguous or linear, presence of lesions in the cervical and thoracic regions, and involvement of the central gray matter or holocord regions on axial scans, were also significantly associated with seropositivity; P<0.001. Conclusion. NMO and HRS for NMOSD are present in larger numbers than previously thought in Malaysia. More than 2/3rds are seropositive. Seropositive and seronegative NMO/NMOSD have differences that are useful in clinical practice.

  13. Abscisic Acid Regulation of Root Hydraulic Conductivity and Aquaporin Gene Expression Is Crucial to the Plant Shoot Growth Enhancement Caused by Rhizosphere Humic Acids.

    Science.gov (United States)

    Olaetxea, Maite; Mora, Verónica; Bacaicoa, Eva; Garnica, María; Fuentes, Marta; Casanova, Esther; Zamarreño, Angel M; Iriarte, Juan C; Etayo, David; Ederra, Iñigo; Gonzalo, Ramón; Baigorri, Roberto; García-Mina, Jose M

    2015-12-01

    The physiological and metabolic mechanisms behind the humic acid-mediated plant growth enhancement are discussed in detail. Experiments using cucumber (Cucumis sativus) plants show that the shoot growth enhancement caused by a structurally well-characterized humic acid with sedimentary origin is functionally associated with significant increases in abscisic acid (ABA) root concentration and root hydraulic conductivity. Complementary experiments involving a blocking agent of cell wall pores and water root transport (polyethylenglycol) show that increases in root hydraulic conductivity are essential in the shoot growth-promoting action of the model humic acid. Further experiments involving an inhibitor of ABA biosynthesis in root and shoot (fluridone) show that the humic acid-mediated enhancement of both root hydraulic conductivity and shoot growth depended on ABA signaling pathways. These experiments also show that a significant increase in the gene expression of the main root plasma membrane aquaporins is associated with the increase of root hydraulic conductivity caused by the model humic acid. Finally, experimental data suggest that all of these actions of model humic acid on root functionality, which are linked to its beneficial action on plant shoot growth, are likely related to the conformational structure of humic acid in solution and its interaction with the cell wall at the root surface.

  14. Cloning and characterization of porcine aquaporin 1 water channel expressed extensively in gastrointestinal system

    Institute of Scientific and Technical Information of China (English)

    Shun-Ying Jin; Yan-Li Liu; Li-Na Xu; Yong Jiang; Ying Wang; Bao-Xue Yang; Hong Yang; Tong-Hui Ma

    2006-01-01

    AIM: To clone and characterize the porcine aquaporins (AQPs) in the gastrointestinal system.METHODS: A PCR-based cloning strategy and RACE were used to clone full-length AQP coding sequence from reversely transcribed pig liver cDNA. Stopped-flow light scattering and a YFP-based fluorescence method were used to measure the osmotic water permeability of erythrocytes and the stably transfected CHO cells.RT-PCR, Northern blot, and immunohistochemistry were used to determine the gastrointestinal expression and localization of cloned AQPs. Protein expression in transfected cells and red blood cells was analyzed by Western blot.RESULTS: An 813 bp cDNA encoding a 271 amino acid porcine aquaporin (designated pAQP1) was cloned from liver mRNA (pAQP1 has a 93% identity with human AQP1 and contains two NPA motifs conserved in AQP family, one consensus sequence for N-linked glycosylation, and one mercury-sensitive site at cysteine 191). RT-PCR analysis revealed extensive expression of pAQP1 mRNA in porcine digestive glands and gut.Northern blot showed a single 3.0 kb transcript in selected digestive organs, pAQP1 protein was localized at central lacteals of the small intestine, microvessles of salivary glands, as well as epithelium of intrahepatic bile ducts by immunoperoxydase. High osmotic water permeability that is inhibitable by HgCl2 was detected in porcine erythrocytes and CHO cells stably transfected with pAQP1 cDNA. Tmmunoblot analysis of porcine erythrocytes and pAQP-transfected CHO cells revealed an unglycosylated 28 ku band and larger glycosylated proteins.CONCLUSION: pAQP1 is the first porcine aquaporin that can be molecularly identified so far. The broad distribution of pAQP1 in epithelium and endothelium of porcine digestive organs may suggest an important role of channel-mediated water transport in fluid secretion/absorption as well as in digestive function and pathophysiology of the gastrointestinal system.

  15. Aquaporin 4 antibody [NMO Ab] status in patients with severe optic neuritis in India.

    Science.gov (United States)

    Ambika, Selvakumar; Balasubramanian, Mahalakshmi; Theresa, Lily; Veeraputhiran, Akila; Arjundas, Deepak

    2015-12-01

    Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system that causes attacks of optic neuritis and transverse myelitis. The discovery of a specific serum marker for NMO-IgG antibody [aquaporin 4 antibody/AQP4 Ab] has revolutionised the treatment of demyelinating diseases. Severe vision loss can be seen in optic neuritis (ON) associated with both multiple sclerosis (MS) and NMO. Identifying this antibody in optic neuritis patients can help us to establish the likelihood of these patients developing NMO (Jarius et al. Neurol Sci 298:158-162, 2010). It is important to differentiate these two entities as the treatment strategies of MS and NMO are different. To the best of our knowledge, there is no published literature regarding the importance of identifying this antibody in severe optic neuritis in Indian patients. Hence we decided to screen our severe optic neuritis patients for this AQP4 Ab. To investigate the presence of aquaporin 4 antibody and determine its prognostic value for visual and neurological outcome, in patients with bilateral and recurrent [severe] ON without any previous neurological manifestations presenting to a neuro-ophthalmology clinic in India. Single centre, prospective study. 40 patients (27 female patients and 13 male) with severe optic neuritis [patients with no visual improvement by 4 weeks from onset of vision loss] who presented either as recurrent attacks or as bilateral and severe optic neuritis between January 2010 and June 2011 were enrolled. Clinical features, visual outcome and sequential neurological events were compared between the seropositive and the seronegative groups. Aquaporin 4 antibodies were detected from serum using ELISA technique and IIF technique. Presence of this antibody in the serum was considered to be seropositive status and patients who did not have this antibody were considered seronegatives. AQP4 antibodies were detected in 8 of the 40 patients with severe ON (20 %).The

  16. Preventive administration of cromakalim reduces aquaporin-4 expression and blood-brain barrier permeability in a rat model of cerebral ischemia/reperfusion injury

    Institute of Scientific and Technical Information of China (English)

    Shilei Wang; Yanting Wang; Yan Jiang; Qingxian Chang; Peng Wang; Shiduan Wang

    2011-01-01

    Cromakalim, an adenosine triphosphate-sensitive potassium channel opener, exhibits protective effects on cerebral ischemia/reperfusion injury. However, there is controversy as to whether this effect is associated with aquaporin-4 and blood-brain barrier permeability. Immunohistochemistry results show that preventive administration of cromakalim decreased aquaporin-4 and IgG protein expression in rats with ischemia/reperfusion injury; it also reduced blood-brain barrier permeability, and alleviated brain edema, ultimately providing neuroprotection.

  17. Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Boesen, Jane; Karlsen, Pernille Efferbach;

    2009-01-01

    Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed...

  18. Identification of cardiac myofilament protein isoforms using multiple mass spectrometry based approaches

    NARCIS (Netherlands)

    Kooij, V.; Venkatraman, V.; Kirk, J.A.; Ubaida-Mohien, C.; Graham, D.R.; Faber, M.J.; Eyk, J.E. Van

    2014-01-01

    PURPOSE: The identification of protein isoforms in complex biological samples is challenging. We, therefore, used an MS approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform. EXPER

  19. Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors

    DEFF Research Database (Denmark)

    Khan, N.; Jeffers, M.; Kumar, S.;

    2008-01-01

    ) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rh...

  20. A chemokine targets the nucleus: Cxcl12-gamma isoform localizes to the nucleolus in adult mouse heart.

    Directory of Open Access Journals (Sweden)

    Raul Torres

    Full Text Available Chemokines are extracellular mediators of complex regulatory circuits involved principally in cell-to-cell communication. Most studies to date of the essential chemokine Cxcl12 (Sdf-1 have focused on the ubiquitously expressed secreted isoforms alpha and beta. Here we show that, unlike these isoforms and all other known chemokines, the alternatively transcribed gamma isoform is an intracellular protein that localizes to the nucleolus in differentiated mouse Cardiac tissue. Our results demonstrate that nucleolar transportation is encoded by a nucleolar-localization signal in the unique carboxy-terminal region of Sdf-1gamma, and is competent both in vivo and in vitro. The molecular mechanism underlying these unusual chemokine properties involves cardiac-specific transcription of an mRNA containing a unique short-leader sequence lacking the signal peptide and translation from a non-canonical CUG codon. Our results provide an example of genome economy even for essential and highly conserved genes such as Cxcl12, and suggest that chemokines can exert tissue specific functions unrelated to cell-to-cell communication.

  1. ADP-ribosylhydrolase 3 (ARH3), Not Poly(ADP-ribose) Glycohydrolase (PARG) Isoforms, Is Responsible for Degradation of Mitochondrial Matrix-associated Poly(ADP-ribose)*

    Science.gov (United States)

    Niere, Marc; Mashimo, Masato; Agledal, Line; Dölle, Christian; Kasamatsu, Atsushi; Kato, Jiro; Moss, Joel; Ziegler, Mathias

    2012-01-01

    Important cellular processes are regulated by poly(ADP-ribosyl)ation. This protein modification is catalyzed mainly by nuclear poly(ADP-ribose) polymerase (PARP) 1 in response to DNA damage. Cytosolic PARP isoforms have been described, whereas the presence of poly(ADP-ribose) (PAR) metabolism in mitochondria is controversial. PAR is degraded by poly(ADP-ribose) glycohydrolase (PARG). Recently, ADP-ribosylhydrolase 3 (ARH3) was also shown to catalyze PAR-degradation in vitro. PARG is encoded by a single, essential gene. One nuclear and three cytosolic isoforms result from alternative splicing. The presence and origin of a mitochondrial PARG is still unresolved. We establish here the genetic background of a human mitochondrial PARG isoform and investigate the molecular basis for mitochondrial poly(ADP-ribose) degradation. In common with a cytosolic 60-kDa human PARG isoform, the mitochondrial protein did not catalyze PAR degradation because of the absence of exon 5-encoded residues. In mice, we identified a transcript encoding an inactive cytosolic 52-kDa PARG lacking the mitochondrial targeting sequence and a substantial portion of exon 5. Thus, mammalian PARG genes encode isoforms that do not catalyze PAR degradation. On the other hand, embryonic fibroblasts from ARH3−/− mice lack most of the mitochondrial PAR degrading activity detected in wild-type cells, demonstrating a potential involvement of ARH3 in PAR metabolism. PMID:22433848

  2. ADP-ribosylhydrolase 3 (ARH3), not poly(ADP-ribose) glycohydrolase (PARG) isoforms, is responsible for degradation of mitochondrial matrix-associated poly(ADP-ribose).

    Science.gov (United States)

    Niere, Marc; Mashimo, Masato; Agledal, Line; Dölle, Christian; Kasamatsu, Atsushi; Kato, Jiro; Moss, Joel; Ziegler, Mathias

    2012-05-11

    Important cellular processes are regulated by poly(ADP-ribosyl)ation. This protein modification is catalyzed mainly by nuclear poly(ADP-ribose) polymerase (PARP) 1 in response to DNA damage. Cytosolic PARP isoforms have been described, whereas the presence of poly(ADP-ribose) (PAR) metabolism in mitochondria is controversial. PAR is degraded by poly(ADP-ribose) glycohydrolase (PARG). Recently, ADP-ribosylhydrolase 3 (ARH3) was also shown to catalyze PAR-degradation in vitro. PARG is encoded by a single, essential gene. One nuclear and three cytosolic isoforms result from alternative splicing. The presence and origin of a mitochondrial PARG is still unresolved. We establish here the genetic background of a human mitochondrial PARG isoform and investigate the molecular basis for mitochondrial poly(ADP-ribose) degradation. In common with a cytosolic 60-kDa human PARG isoform, the mitochondrial protein did not catalyze PAR degradation because of the absence of exon 5-encoded residues. In mice, we identified a transcript encoding an inactive cytosolic 52-kDa PARG lacking the mitochondrial targeting sequence and a substantial portion of exon 5. Thus, mammalian PARG genes encode isoforms that do not catalyze PAR degradation. On the other hand, embryonic fibroblasts from ARH3(-/-) mice lack most of the mitochondrial PAR degrading activity detected in wild-type cells, demonstrating a potential involvement of ARH3 in PAR metabolism.

  3. Tolerance to the antinociceptive and hypothermic effects of morphine is mediated by multiple isoforms of c-Jun N-terminal kinase.

    Science.gov (United States)

    Yuill, Matthew B; Zee, Michael L; Marcus, David; Morgan, Daniel J

    2016-04-13

    The abuse and overdose of opioid drugs are growing public health problems worldwide. Although progress has been made toward understanding the mechanisms governing tolerance to opioids, the exact cellular machinery involved remains unclear. However, there is growing evidence to suggest that c-Jun N-terminal kinases (JNKs) play a major role in mu-opioid receptor regulation and morphine tolerance. In this study, we aimed to determine the potential roles of different JNK isoforms in the development of tolerance to the antinociceptive and hypothermic effects of morphine. We used the hot-plate and tail-flick tests for thermal pain to measure tolerance to the antinociceptive effects of once-daily subcutaneous injections with 10 mg/kg morphine. Body temperature was also measured to determine tolerance to the hypothermic effects of morphine. Tolerance to morphine was assessed in wild-type mice and compared with single knockout mice each lacking the JNK isoforms (JNK1, JNK2, or JNK3). We found that loss of each individual JNK isoform causes impairment in tolerance for the antinociceptive and hypothermic effects of daily morphine. However, disruption of JNK2 seems to have the most profound effect on morphine tolerance. These results indicate a clear role for JNK signaling pathways in morphine tolerance. This complements previous studies suggesting that the JNK2 isoform is required for morphine tolerance, but additionally presents novel data suggesting that additional JNK isoforms also contribute toward this process.

  4. Sponge biosilica formation involves syneresis following polycondensation in vivo.

    Science.gov (United States)

    Wang, Xiaohong; Schröder, Heinz C; Brandt, David; Wiens, Matthias; Lieberwirth, Ingo; Glasser, Gunnar; Schlossmacher, Ute; Wang, Shunfeng; Müller, Werner E G

    2011-10-17

    Syneresis is a process observed during the maturation/aging of silica gels obtained by sol-gel synthesis that results in shrinkage and expulsion of water due to a rearrangement and increase in the number of bridging siloxane bonds. Here we describe how the process of biosilica deposition during spicule ("biosilica" skeleton of the siliceous sponges) formation involves a phase of syneresis that occurs after the enzyme-mediated polycondensation reaction. Primmorphs from the demosponge Suberites domuncula were used to study syneresis and the inhibition of this mechanism. We showed by scanning electron microscopy that spicules added to primmorphs that have been incubated with manganese sulfate fuse together through the deposition of silica spheres and bridges. Energy-dispersive X-ray mapping of the newly formed deposits showed high silicon and oxygen content. These biosilica deposits contain a comparably higher percentage of water than mature/aged spicules. Quantitative real-time polymerase chain reaction analyses revealed that the addition of silicate to primmorph cultures resulted in a marked upregulation of the expression of the aquaporin gene and of the genes encoding the silica anabolic enzyme silicatein-α and the silica catabolic enzyme silicase. On the other hand, addition of manganese sulfate, either alone or together with silicate, caused a strong reduction in the level of aquaporin transcripts, although this metal ion did not essentially affect the silicate-induced increase in silicatein-α and silicase gene expression. We conclude that the secondary silica deposits formed on spicules under physiological conditions in the presence of silicate fuse together and subsequently undergo syneresis, which is facilitated by the removal of water through aquaporin channels. In growing spicules, these processes of biosilica formation and syneresis in the lamellar monolithic structures precede the final step of "biosintering" during which the massive biosilica rods of

  5. Demeclocycline Attenuates Hyponatremia by Reducing Aquaporin-2 Expression in the Renal Inner Medulla

    DEFF Research Database (Denmark)

    Kortenoeven, Marleen L. A.; Sinke, Anne P.; Hadrup, Niels;

    2013-01-01

    Binding of vasopressin to its type-2 receptor in renal collecting ducts induces cAMP signaling, transcription and translocation of aquaporin-2 (AQP2) water channels to the plasma membrane and water reabsorption from the pro-urine. Demeclocycline is currently used to treat hyponatremia in patients...... urine volume, decreased urine osmolality and reverted hyponatremia in an SIADH rat model. AQP2 and adenylate cyclase 5/6 abundances were reduced in the inner medulla, but increased in the cortex and outer medulla, in the absence of any sign of toxicity. In conclusion, our in vitro and in vivo data...... indicate that demeclocycline mainly attenuates hyponatremia in SIADH by reducing adenylate cyclase 5/6 expression, and consequently cAMP generation, AQP2 gene transcription and AQP2 abundance in the renal inner medulla, coinciding with a reduced vasopressin-escape response in the other collecting duct...

  6. Immunohistochemical Study of Aquaporins in an African Grey Parrot (Psittacus erithacus) With Hydrocephalus.

    Science.gov (United States)

    Blasco, Ester; Martorell, Jaime; De la Fuente, Cristian; Pumarola, Martí

    2014-12-01

    A 5-month-old African grey parrot (Psittacus erithacus) was examined after 3 weeks of weakness, ataxia, mental depression, and seizures. Results of a complete blood cell count and plasma biochemical analysis were unremarkable. Magnetic resonance imaging revealed a severe bilateral hydrocephalus. The bird failed to improve with supportive care, and the owner requested euthanasia. Necropsy findings were severe bilateral hydrocephalus with no evidence of cerebrospinal fluid obstruction. Histologic examination of the brain revealed microspongiosis, edema, gliosis, and neuronal chromatolysis of surrounding periventricular tissue. Aquaporins (AQP) and astrocytes were examined to elucidate the participation of these water channel proteins and glial cells in the pathophysiology and resolution of hydrocephalus. Results showed AQP4 and glial fibrillary acidic protein were overexpressed, especially near the ventricles, but expression of AQP1 was decreased. This is the first report, to our knowledge, of AQP immunolabeling in hydrocephalus in avain species.

  7. Simple and inexpensive hardware and software method to measure volume changes in Xenopus oocytes expressing aquaporins.

    Science.gov (United States)

    Dorr, Ricardo; Ozu, Marcelo; Parisi, Mario

    2007-04-15

    Water channels (aquaporins) family members have been identified in central nervous system cells. A classic method to measure membrane water permeability and its regulation is to capture and analyse images of Xenopus laevis oocytes expressing them. Laboratories dedicated to the analysis of motion images usually have powerful equipment valued in thousands of dollars. However, some scientists consider that new approaches are needed to reduce costs in scientific labs, especially in developing countries. The objective of this work is to share a very low-cost hardware and software setup based on a well-selected webcam, a hand-made adapter to a microscope and the use of free software to measure membrane water permeability in Xenopus oocytes. One of the main purposes of this setup is to maintain a high level of quality in images obtained at brief intervals (shorter than 70 ms). The presented setup helps to economize without sacrificing image analysis requirements.

  8. Reduced Bone Mineral Density and Bone Metabolism in Aquaporin-1 Knockout Mice

    Institute of Scientific and Technical Information of China (English)

    WU Qing-tian; MA Qing-jie; HE Cheng-yan; WANG Cai-xia; GAO Shi; HOU Xia; MA Tong-hui

    2007-01-01

    An overt phenotype of aquaporin-1 knockout(AQP1 ko) mice is growth retardation, suggesting possible defects in bone development and metabolism. In the present study, we analyzed the bone mineral density(BMD), bone calcium and phosphorus contents, and bone metabolism in an AQP1 ko mouse model. The BMD of femurs in AQP1 ko mice was significantly lower than that of litter-matched wildtype mice as measured by dual energy X-ray absorptiometry. Consistently, the contents of bone total calcium and phosphorus were also significantly lower in AQP1 ko mice. The reduced BMD caused by AQP1 deficiency mainly affect male mice. Bone metabolic activity, as indicated by 99mTc-MDP absorption measurements, was remarkably reduced in AQP1 ko mice. These results provide the first evidence that AQP1 play an important role in bone structure and metabolism.

  9. Bundle-sheath aquaporins play a role in controlling Arabidopsis leaf hydraulic conductivity.

    Science.gov (United States)

    Sade, Nir; Shatil-Cohen, Arava; Moshelion, Menachem

    2015-01-01

    The role of molecular mechanisms in the regulation of leaf hydraulics (K(leaf)) is still not well understood. We hypothesized that aquaporins (AQPs) in the bundle sheath may regulate K(leaf). To examine this hypothesis, AQP genes were constitutively silenced using artificial microRNAs and recovery was achieved by targeting the expression of the tobacco AQP (NtAQP1) to bundle-sheath cells in the silenced plants. Constitutively silenced PIP1 plants exhibited decreased PIP1 transcript levels and decreased K(leaf). However, once the plants were recovered with NtAQP1, their K(leaf) values were almost the same as those of WT plants. We also demonstrate the important role of ABA, acting via AQP, in that recovery and K(leaf) regulation. These results support our previously raised hypothesis concerning the role of bundle-sheath AQPs in the regulation of leaf hydraulics.

  10. Comparative Molecular Docking Studies with ABCC1 and Aquaporin 9 in the Arsenite Complex Efflux

    Science.gov (United States)

    Poojan, Shiv; Dhasmana, Anupam; Jamal, Qazi Mohammad Sajid; Haneef, Mohd; Lohani, Mohtashim

    2014-01-01

    Arsenic is the most toxic metalloid present in the natural environment in both organic and inorganic arsenic forms. Inorganic arsenic is often more hazardous than the organic form. Arsenite and arsenate compounds are the major inorganic forms which are toxic causing severe human health dysfunction including cancer. Excretion of arsenic from the system is found elusive. Therefore, it is of interest to screen channel proteins with the arsenic complex in the different combination of arsenic, GSH (glutathione) and arsenic, selenium using docking methods. The mode of arsenic removal. The complex structure revealed the mode of arsenic binding efficiency with the receptor aquaporine 9 and ABCC1 channel protein. This provides insights to understand the mechanism of arsenic efflux. These inferences find application in the design, identification and development of novel nutracetucal or any other formulation useful in the balance of arsenic efflux. PMID:25258480

  11. Role of aquaporin 9 in cellular accumulation of arsenic and its cytotoxicity in primary mouse hepatocytes.

    Science.gov (United States)

    Shinkai, Yasuhiro; Sumi, Daigo; Toyama, Takashi; Kaji, Toshiyuki; Kumagai, Yoshito

    2009-06-01

    Aquaporin (AQP) 9 is a member of the aquaglyceroporin subfamily of AQPs in the transfer of water and small solutes such as glycerol and arsenite. It is well recognized that arsenic toxicity is associated with intracellular accumulation of this metalloid. In the present study, we examined the contribution of AQP9 to the uptake of inorganic arsenite, thereby increasing arsenic-induced cytotoxicity in primary mouse hepatocytes. Pretreatment with sorbitol as a competitive inhibitor of AQP9 and siRNA-mediated knockdown of AQP9 resulted in a significant decrease of arsenite uptake in the cell and its cytotoxicity. Furthermore, overexpression of AQP9 in HEK293 cells led to the enhancement of intracellular arsenic concentration, resulting in enhanced cytotoxicity after arsenite exposure. These results suggest that AQP9 is a channel to define arsenite sensitivity in primary mouse hepatocytes.

  12. Comparative Molecular Docking Studies with ABCC1 and Aquaporin 9 in the Arsenite Complex Efflux.

    Science.gov (United States)

    Poojan, Shiv; Dhasmana, Anupam; Jamal, Qazi Mohammad Sajid; Haneef, Mohd; Lohani, Mohtashim

    2014-01-01

    Arsenic is the most toxic metalloid present in the natural environment in both organic and inorganic arsenic forms. Inorganic arsenic is often more hazardous than the organic form. Arsenite and arsenate compounds are the major inorganic forms which are toxic causing severe human health dysfunction including cancer. Excretion of arsenic from the system is found elusive. Therefore, it is of interest to screen channel proteins with the arsenic complex in the different combination of arsenic, GSH (glutathione) and arsenic, selenium using docking methods. The mode of arsenic removal. The complex structure revealed the mode of arsenic binding efficiency with the receptor aquaporine 9 and ABCC1 channel protein. This provides insights to understand the mechanism of arsenic efflux. These inferences find application in the design, identification and development of novel nutracetucal or any other formulation useful in the balance of arsenic efflux.

  13. Expression patterns of genes encoding plasma membrane aquaporins during fruit development in cucumber (Cucumis sativus L.).

    Science.gov (United States)

    Shi, Jin; Wang, Jinfang; Li, Ren; Li, Dianbo; Xu, Fengfeng; Sun, Qianqian; Zhao, Bin; Mao, Ai-Jun; Guo, Yang-Dong

    2015-11-01

    Aquaporins are membrane channels precisely regulating water movement through cell membranes in most living organisms. Despite the advances in the physiology of fruit development, their participation during fruit development in cucumber still barely understood. In this paper, the expressions of 12 genes encoding plasma membrane intrinsic proteins (PIPs) were analyzed during cucumber fruit development in our work. Based on the homology search with known PIPs from rice, Arabidopsis and strawberry, 12 cucumber PIP genes subfamily members were identified. Cellular localization assays indicated that CsPIPs were localized in the plasma membrane. The qRT-PCR analysis of CsPIPs showed that 12 CsPIPs were differentially expressed during fruit development. These results suggest that 12 genes encoding plasma membrane intrinsic proteins (CsPIPs) play very important roles in cucumber life cycle and the data generated will be helpful in understanding their precise roles during fruit development in cucumber.

  14. Regional expression of aquaporin 1, 4, and 9 in the brain during pregnancy.

    Science.gov (United States)

    Wiegman, Marchien J; Bullinger, Lisa V; Kohlmeyer, Meghan M; Hunter, Timothy C; Cipolla, Marilyn J

    2008-05-01

    Pregnancy is a state of physiologic adaptation, with significant changes in cardiovascular, renal, and hemodynamic systems. Aquaporins (AQPs) may play a role in facilitating these changes. While AQP expression has been assessed in several organs during pregnancy, little is known about its expression in the brain during pregnancy. Therefore, this study assesses the regional expression of AQP1, 4, and 9 during pregnancy and the postpartum period using real-time quantitative polymerase chain reaction. The authors show that AQP1, 4, and 9 are expressed in the anterior and posterior cerebrum, cerebellum, and brainstem of nonpregnant, midpregnant, late pregnant, and postpartum rats. The regional distribution pattern of AQP4 and 9 remained similar during gestation, whereas this pattern changed for AQP1. The expression levels of AQP1, 4, and 9 in the brainstem did not change with gestation, whereas changes were found in the anterior cerebrum for AQP4 and in the posterior cerebrum and cerebellum for all AQPs.

  15. A novel role for aquaporin-5 in enhancing microtubule organization and stability.

    Directory of Open Access Journals (Sweden)

    Venkataramana K Sidhaye

    Full Text Available Aquaporin-5 (AQP5 is a water-specific channel located on the apical surface of airway epithelial cells. In addition to regulating transcellular water permeability, AQP5 can regulate paracellular permeability, though the mechanisms by which this occurs have not been determined. Microtubules also regulate paracellular permeability. Here, we report that AQP5 promotes microtubule assembly and helps maintain the assembled microtubule steady state levels with slower turnover dynamics in cells. Specifically, reduced levels of AQP5 correlated with lower levels of assembled microtubules and decreased paracellular permeability. In contrast, overexpression of AQP5 increased assembly of microtubules, with evidence of increased MT stability, and promoted the formation of long straight microtubules in the apical domain of the epithelial cells. These findings indicate that AQP5-mediated regulation of microtubule dynamics modulates airway epithelial barrier properties and epithelial function.

  16. Tubular localization and expressional dynamics of aquaporins in the kidney of seawater-challenged Atlantic salmon

    DEFF Research Database (Denmark)

    Engelund, Morten Buch; Madsen, Steffen S

    2015-01-01

    . Aqp10b was expressed apically and along the lateral membrane. Aqp8b was mainly basolateral and Aqp1ab was located in sub-apical intracellular compartments. mRNAs of aqp8b and aqp10b were higher in FW smolts compared to FW parr, whereas the opposite was true for aqp1aa. Aqp mRNA levels changed......10b were localized in the kidney of salmon. The staining for all aquaporins was most abundant in the proximal kidney tubules and there was no clear effect of salinity or developmental stage on localization pattern. Aqp1aa and Aqp3a were abundant apically but extended throughout the epithelial cells...

  17. Increased expression of aquaporin-4 in brain tissue of amygdala-kindled rats

    Institute of Scientific and Technical Information of China (English)

    Yinghui Chen; Yongbo Zhao

    2011-01-01

    Recurrent epileptic seizures can lead to brain edema, indicating that water regulation may be perturbed by seizures.We hypothesized that the expression of the brain water channel aquaporin-4 (AQP-4) may be upregulated in the epileptic brain.In the present study, we established the amygdala kindling model of epilepsy, and quantified AQP-4 protein and mRNA levels, using reverse transcription-PCR, immunohistochemistry and western blotting, in epileptic and control rats.We found that AQP-4 was overexpressed in the cerebral cortex of rats with epilepsy compared with controls.These findings show that AQP-4 is highly expressed in the brain of amygdala-kindled rats, suggesting that repeated seizures affect water homeostasis in the brain.

  18. Neuromyelitis optica-IgG (aquaporin-4) autoantibodies in immune mediated optic neuritis.

    Science.gov (United States)

    Petzold, A; Pittock, S; Lennon, V; Maggiore, C; Weinshenker, B G; Plant, G T

    2010-01-01

    The clinical course of immune mediated optic neuritis (ON) will depend on the specific underlying inflammatory disease. These disorders have traditionally been classified according to clinical and MRI findings. Aquaporin-4 (AQP4) autoantibodies (neuromyelitis optica-IgG (NMO-IgG)) may have diagnostic and prognostic value in patients who present with isolated ON. In this prospective study, NMO-IgG was evaluated in 114 patients with ON in the following contexts: neuromyelitis optica (NMO), multiple sclerosis (MSON), chronic relapsing inflammatory ON (CRION), relapsing isolated ON (RION) and single isolated ON (SION). The proportion seropositive was 56% for NMO (n = 9), 0% for MSON (n = 28) and 5% for the remaining diagnostic categories (CRION (n = 19), RION (n = 17) and SION (n = 41)). Testing for NMO-IgG in patients with recurrent or severe ON who lack convincing evidence of MS may identify patients who would benefit from immunosuppression rather than MS directed immunomodulatory therapies.

  19. Differential regulation of macropinocytosis by Abi1/Hssh3bp1 isoforms.

    Directory of Open Access Journals (Sweden)

    Patrycja M Dubielecka

    Full Text Available BACKGROUND: Macropinocytosis, which is a constitutive cellular process of fluid and macromolecule uptake, is regulated by actin cytoskeleton rearrangements near the plasma membrane. Activation of Rac1, which is proposed to act upstream of the actin polymerization regulatory Wave 2 complex, has been found to correlate with enhanced macropinocytosis. One of the components of the Wave 2 complex is Abi1. Multiple, alternatively spliced isoforms of Abi1 are expressed in mammalian cells, but the functional significance of the various isoforms is unknown. PRINCIPAL FINDINGS: Here, using flow cytometric assay analysis for Alexa Fluor 647, we demonstrate that Abi1 isoforms 2 and 3 differentially regulate macropinocytosis. LNCaP cells expressing isoform 3 had increased macropinocytic uptake that correlated with enhanced cell spreading and higher Rac1 activation in comparison to cells expressing isoform 2. Isoform 2 expressing cells had decreased macropinocytic uptake, but demonstrated greater sensitivity to Rac1 activation. Moreover, more isoform 2 was localized within the cytoplasm in comparison to isoform 3, which was more associated with the plasma membrane. Activated Rac1 was found to specifically bind to a site in exon 10 of isoform 2 in vitro. Because of alternative mRNA splicing, exon 10 is absent from isoform 3, precluding similar binding of activated Rac1. Both isoforms, however, bound to inactive Rac1 through the same non-exon 10 site. Thus, Abi1 isoform 3-containing Wave 2 complex exhibited a differential binding to activated vs. inactive Rac1, whereas isoform 2-containing Wave 2 complex bound activated or inactive Rac1 comparably. CONCLUSION: Based on these observations, we postulate that Abi1 isoforms differentially regulate macropinocytosis as a consequence of their different relative affinities for activated Rac1 in Wave 2 complex. These findings also raise the possibility that isoform-specific roles occur in other Abi1 functions.

  20. Distribution and quantitative changes in amounts of aquaporin 1, 5 and 9 in the pig uterus during the estrous cycle and early pregnancy

    Directory of Open Access Journals (Sweden)

    Skowronski Mariusz T

    2010-09-01

    Full Text Available Abstract Background Aquaporins (AQPs are a family of membrane channel proteins that facilitate bulk water transport. To date, 11 isoforms of AQPs have been reported to be expressed in the female and male reproductive systems. The purpose of our study was to determine the localization and quantitative changes in the expression of AQP1, 5 and 9 within the pig uterus during different stages of the estrous cycle and early pregnancy. Methods Immunoperoxidase and semi-quantitative immunoblotting techniques were used to examine the distribution and changes in amounts of AQP1, AQP5 and AQP9 in uteral cells of pigs at the early (Days 2-4, middle (10-12, late (14-16 stage of the luteal phase and late (18-20 stage of the follicular phase of the estrous cycle as well as on Days 14-16 and 30-32 of gestation (the onset and the end of implantation process. Results The results demonstrated that AQP1, 5, and 9 were clearly detected in all studied stages of the estrous cycle and pregnancy. AQP1 was localized within uterine blood vessels. In cyclic gilts, endometrial and myometrial expression of AQP1 protein did not change significantly but increased during gestation. AQP5 was localized in smooth muscle cells and uterine epithelial cells. Endometrial expression of AQP5 protein did not change significantly between Days 2-4 and 10-12 of the estrous cycle but increased on Days 14-16 and 18-20 as well as during early pregnancy. Myometrial expression of AQP5 did not differ significantly during the estrous cycle but increased in the pregnancy. The anti-AQP9 antibody labeled uterine epithelial cells of uterus. Endometrial expression of AQP9 did not change significantly between Days 2-4 and 10-12 of the estrous cycle but increased on Days 14-16 and 18-20 as well as during early pregnancy. Conclusions The results suggest that a functional and distinctive collaboration exists among diverse AQPs in water handling during the different uterine phases in the estrous cycle and

  1. Molecular cloning and characterisation of two calmodulin isoforms of the Madagascar periwinkle Catharanthus roseus.

    Science.gov (United States)

    Poutrain, P; Guirimand, G; Mahroug, S; Burlat, V; Melin, C; Ginis, O; Oudin, A; Giglioli-Guivarc'h, N; Pichon, O; Courdavault, V

    2011-01-01

    Involvement of Ca(2+) signalling in regulation of the biosynthesis of monoterpene indole alkaloids (MIA) in Catharanthus roseus has been extensively studied in recent years, albeit no protein of this signalling pathway has been isolated. Using a PCR strategy, two C. roseus cDNAs encoding distinct calmodulin (CAM) isoforms were cloned and named CAM1 and CAM2. The deduced 149 amino acid sequences possess four Ca(2+) binding domains and exhibit a close identity with Arabidopsis CAM isoforms (>91%). The ability of CAM1 and CAM2 to bind Ca(2+) was demonstrated following expression of the corresponding recombinant proteins. Furthermore, transient expression of CAM1-GFP and CAM2-GFP in C. roseus cells showed a typical nucleo-cytoplasm localisation of both CAMs, in agreement with the wide distribution of CAM target proteins. Using RNA blot analysis, we showed that CAM1 and CAM2 genes had a broad pattern of expression in C. roseus organs and are constitutively expressed during a C. roseus cell culture cycle, with a slight inhibitory effect of auxin for CAM1. Using RNA in situ hybridisation, we also detected CAM1 and CAM2 mRNA in the vascular bundle region of young seedling cotyledons. Finally, using specific inhibitors, we also showed that CAMs are required for MIA biosynthesis in C. roseus cells by acting on regulation of expression of genes encoding enzymes that catalyse early steps of MIA biosynthesis, such as 1-deoxy-d-xylulose 5-phosphate reductoisomerase and geraniol 10-hydroxylase.

  2. Somatodendritic and excitatory postsynaptic distribution of neuron-type dystrophin isoform, Dp40, in hippocampal neurons.

    Science.gov (United States)

    Fujimoto, Takahiro; Itoh, Kyoko; Yaoi, Takeshi; Fushiki, Shinji

    2014-09-12

    The Duchenne muscular dystrophy (DMD) gene produces multiple dystrophin (Dp) products due to the presence of several promoters. We previously reported the existence of a novel short isoform of Dp, Dp40, in adult mouse brain. However, the exact biochemical expression profile and cytological distribution of the Dp40 protein remain unknown. In this study, we generated a polyclonal antibody against the NH2-terminal region of the Dp40 and identified the expression profile of Dp40 in the mouse brain. Through an analysis using embryonic and postnatal mouse cerebrums, we found that Dp40 emerged from the early neonatal stages until adulthood, whereas Dp71, an another Dp short isoform, was highly detected in both prenatal and postnatal cerebrums. Intriguingly, relative expressions of Dp40 and Dp71 were prominent in cultured dissociated neurons and non-neuronal cells derived from mouse hippocampus, respectively. Furthermore, the immunocytological distribution of Dp40 was analyzed in dissociated cultured neurons, revealing that Dp40 is detected in the soma and its dendrites, but not in the axon. It is worthy to note that Dp40 is localized along the subplasmalemmal region of the dendritic shafts, as well as at excitatory postsynaptic sites. Thus, Dp40 was identified as a neuron-type Dp possibly involving dendritic and synaptic functions.

  3. Interaction between oblongifolin C and UDP-glucuronosyltransferase isoforms in human liver and intestine microsomes.

    Science.gov (United States)

    Gao, Cui; Shi, Rong; Wang, Tianming; Tan, Hongsheng; Xu, Hongxi; Ma, Yueming

    2015-01-01

    1. Oblongifolin C (OC) is a potential natural anticancer candidate, and its metabolic profile has not yet been established. 2. One major OC glucuronidation metabolite (OCG) has been identified in a pool of human liver microsomes (HLMs). Chemical inhibition experiments suggested that OCG was mainly formed by UGT1A. A screen of recombinant UDP-glucuronosyltransferase isoforms (UGTs) indicated that UGT1A1 primarily mediates OC conjugation, with minor contributions from UGT1A3 and UGT1A8. Enzyme kinetic studies showed that UGT1A1 was the main UGT isoform involved in OCG in HLMs. 3. Further investigation suggested that OC is a broad inhibitor of UGTs. Additionally, OC competitively inhibited UGT1A6 with a Ki value of 3.49 ± 0.57 μM, whereas non-competitively inhibited UGT1A10 with a Ki value of 2.12 ± 0.18 μM. 4. Understanding the interaction between OC and UGTs will greatly contribute to future investigations regarding the inter-individual differences in OC metabolism in clinical trials and potential drug-drug interactions.

  4. Roles of Aquaporins in Setaria viridis Stem Development and Sugar Storage

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    Samantha Alison McGaughey

    2016-12-01

    Full Text Available Setaria viridis is a C4 grass used as a model for bioenergy feedstocks. The elongating internodes in developing S. viridis stems grow from an intercalary meristem at the base, and progress acropetally towards fully expanded cells that store sugar. During stem development and maturation, water flow is a driver of cell expansion and sugar delivery. As aquaporin proteins are implicated in regulating water flow we analysed elongating and mature internode transcriptomes to identify putative aquaporin encoding genes that had particularly high transcript levels during the distinct stages of internode cell expansion and maturation. We observed that SvPIP2;1 was highly expressed in internode regions undergoing cell expansion, and SvNIP2;2 was highly expressed in mature sugar accumulating regions. Gene co-expression analysis revealed SvNIP2;2 expression was highly correlated with the expression of five putative sugar transporters expressed in the S. viridis internode. To explore the function of the proteins encoded by SvPIP2;1 and SvNIP2;2 we expressed them in Xenopus laevis oocytes and tested their permeability to water. SvPIP2;1 and SvNIP2;2 functioned as water channels in X. laevis oocytes and their permeability was gated by pH. Our results indicate that SvPIP2;1 may function as a water channel in developing stems undergoing cell expansion and SvNIP2;2 is a candidate for retrieving water and possibly a yet to be determined solute from mature internodes. Future research will investigate whether changing the function of these proteins influences stem growth and sugar yield in S. viridis.

  5. 1,3-propanediol binds deep inside the channel to inhibit water permeation through aquaporins.

    Science.gov (United States)

    Yu, Lili; Rodriguez, Roberto A; Chen, L Laurie; Chen, Liao Y; Perry, George; McHardy, Stanton F; Yeh, Chih-Ko

    2016-02-01

    Aquaporins and aquaglyceroporins (AQPs) are membrane channel proteins responsible for transport of water and for transport of glycerol in addition to water across the cell membrane, respectively. They are expressed throughout the human body and also in other forms of life. Inhibitors of human AQPs have been sought for therapeutic treatment for various medical conditions including hypertension, refractory edema, neurotoxic brain edema, and so forth. Conducting all-atom molecular dynamics simulations, we computed the binding affinity of acetazolamide to human AQP4 that agrees closely with in vitro experiments. Using this validated computational method, we found that 1,3-propanediol (PDO) binds deep inside the AQP4 channel to inhibit that particular aquaporin efficaciously. Furthermore, we used the same method to compute the affinities of PDO binding to four other AQPs and one aquaglyceroporin whose atomic coordinates are available from the protein data bank (PDB). For bovine AQP1, human AQP2, AQP4, AQP5, and Plasmodium falciparum PfAQP whose structures were resolved with high resolution, we obtained definitive predictions on the PDO dissociation constant. For human AQP1 whose PDB coordinates are less accurate, we estimated the dissociation constant with a rather large error bar. Taking into account the fact that PDO is generally recognized as safe by the US FDA, we predict that PDO can be an effective diuretic which directly modulates water flow through the protein channels. It should be free from the serious side effects associated with other diuretics that change the hydro-homeostasis indirectly by altering the osmotic gradients.

  6. Devic’s syndrome in aquaporin-4 antibody negative patient. What we need to know …

    Directory of Open Access Journals (Sweden)

    Nunes, Ana Teresa

    2014-12-01

    Full Text Available [english] Introduction: Neuromyelitis optica (NMO is a severe demyelinating syndrome characterized by optic neuritis (ON and acute myelitis. The NMO spectrum is actually recognized to typically evolve as a relapsing disorder that also includes patients with atypical unilateral ON and those with index events of ON and myelitis occurring weeks or even years apart (Jarius/Wildemann 2013. NMO was previously assumed to be a variant of multiple sclerosis (MS, but the discovery of aquaporin-4 antibodies in patients with neuromyelitis optica has led to this view being revised (Mandler 2006, Barnett/Sutton 2012, Wingerchuk et al. 2007. The cause of the condition is still unknown, but it has been shown that the antibodies bind selectively to a water channel expressed mainly on astrocytes at the blood-brain-barrier, which has an important role in the regulation of brain volume and ion homeostasis. However, there are some patients with NMO that are antibodies negative. The diagnosis is made on the basis of case history, clinical examination, magnetic resonance imaging (MRI of the brain and spinal cord, analysis of cerebrospinal fluid (CSF, visual evoked potentials and a blood test with analysis of aquaporin-4 antibodies (Barnett/Sutton 2012, Wingerchuk et al. 2007, Thornton et al. 2011. This suggests that periodical revisions of established concepts and diagnostic criteria are necessary.Purpose: The authors describe an extremely rare case of neuromyelitis optica and the aim of this paper is to call attention for the cases of NMO whith NMO-IgG negative.Methods: The selected method is a case report.Results: To date the patient showed partial recovery of left eye acuity and improvement of muscle strength of upper and lower limbs and does not show recurrence of the disease.Conclusion: NMO has a distinct clinical, imaging and immunopathological features sufficient to distinguish it from MS. This distinction is essential, because the treatment and the prognosis

  7. Proteomic Validation of Transcript Isoforms, Including Those Assembled from RNA-Seq Data

    DEFF Research Database (Denmark)

    Tay, Aidan P; Pang, Chi Nam Ignatius; Twine, Natalie a;

    2015-01-01

    data, and proteomic analysis of the same sample, can identify protein isoforms. RNA-seq data from human mesenchymal (hMSC) stem cells were analyzed with our new TranscriptCoder tool to generate a database of protein isoform sequences. MS/MS data from matching hMSC samples were then matched against...... the TranscriptCoder-derived database, along with Ensembl and the neXtProt database. Querying the TranscriptCoder-derived or Ensembl database could unambiguously identify ∼450 protein isoforms, with isoform-specific proteotypic peptides, including candidate hMSC-specific isoforms for the genes DPYSL2 and FXR1...

  8. Functional diversity of human basic helix-loop-helix transcription factor TCF4 isoforms generated by alternative 5' exon usage and splicing.

    Directory of Open Access Journals (Sweden)

    Mari Sepp

    Full Text Available BACKGROUND: Transcription factor 4 (TCF4 alias ITF2, E2-2, ME2 or SEF2 is a ubiquitous class A basic helix-loop-helix protein that binds to E-box DNA sequences (CANNTG. While involved in the development and functioning of many different cell types, recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and TCF4 haploinsufficiency is the cause of the Pitt-Hopkins mental retardation syndrome. However, the structure, expression and coding potential of the human TCF4 gene have not been described in detail. PRINCIPAL FINDINGS: In the present study we used human tissue samples to characterize human TCF4 gene structure and TCF4 expression at mRNA and protein level. We report that although widely expressed, human TCF4 mRNA expression is particularly high in the brain. We demonstrate that usage of numerous 5' exons of the human TCF4 gene potentially yields in TCF4 protein isoforms with 18 different N-termini. In addition, the diversity of isoforms is increased by alternative splicing of several internal exons. For functional characterization of TCF4 isoforms, we overexpressed individual isoforms in cultured human cells. Our analysis revealed that subcellular distribution of TCF4 isoforms is differentially regulated: Some isoforms contain a bipartite nuclear localization signal and are exclusively nuclear, whereas distribution of other isoforms relies on heterodimerization partners. Furthermore, the ability of different TCF4 isoforms to regulate E-box controlled reporter gene transcription is varied depending on whether one or both of the two TCF4 transcription activation domains are present in the protein. Both TCF4 activation domains are able to activate transcription independently, but act synergistically in combination. CONCLUSIONS: Altogether, in this study we have described the inter-tissue variability of TCF4 expression in human and provided evidence

  9. Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1

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    Savinska L. O.

    2015-08-01

    Full Text Available Aim. Generation of polyclonal antibodies specific to the ribosomal protein S6 kinase isoform – p85S6K1 and directed to the N-terminal (1–23 aa extension of p85S6K1. Methods. Animal immunization with synthetic (1–23 aa peptide, ELISA, Western blot, Immunoprecipitation, immunofluorescent analysis. Results. Polyclonal antibodies have been generated, which specifically recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofluorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension – the identification of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms.

  10. Disulfide isoforms of recombinant glia maturation factor beta.

    Science.gov (United States)

    Zaheer, A; Lim, R

    1990-09-14

    Recombinant human glia maturation factor beta (r-hGMF-beta) is a single-chain polypeptide (141 amino acid residues) containing three cysteines, at positions 7, 86 and 95. Nascent r-hGMF-beta exists in the reduced state and has no biological activity. The protein can be activated through oxidative refolding by incubation with a mixture of reduced and oxidized glutathione. Reverse-phase HPLC analysis of the refolded r-hGMF-beta shows the presence of four peaks, corresponding to the reduced form plus three newly generated intrachain disulfide-containing isoforms predicted from the number of cysteine residues. Only one isoform shows biological activity when tested for growth suppression on C6 glioma cells. We infer from the HPLC elution pattern that the active form contains the disulfide bridge Cys86-Cys95.

  11. Method for analysing glycoprotein isoforms by capillary electrophoresis

    OpenAIRE

    Frutos, Mercedes de; Díez-Masa, José Carlos; Morales-Cid, Gabriel

    2011-01-01

    [EN] The present invention relates to a new method for the purification, concentration, separation and determination of the isoforms of alpha-1-acid glycoprotein (AGP) in human blood serum samples using capillary electrophoresis. The new method is based on the immunocapture and preconcentration of the sample within the separation capillary by using an immunoadsorbent phase magnetically immobilized within the electrophoresis capillary and the subsequent desorption and separation of the glycopr...

  12. Significance of redox-active cysteines in human FAD synthase isoform 2.

    Science.gov (United States)

    Miccolis, Angelica; Galluccio, Michele; Nitride, Chiara; Giancaspero, Teresa Anna; Ferranti, Pasquale; Iametti, Stefania; Indiveri, Cesare; Bonomi, Francesco; Barile, Maria

    2014-12-01

    FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is the last enzyme in the pathway converting riboflavin into FAD. In humans, FADS is localized in different subcellular compartments and exists in different isoforms. Isoform 2 (490-amino acids) is organized in two domains: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and one resembling a molybdopterin-binding (MPTb) domain, with a hypothetical regulatory role. hFADS2 contains ten Cys residues, seven of which located in the PAPS reductase domain, with a possible involvement either in FAD synthesis or in FAD delivery to cognate apo-flavoproteins. A homology model of the PAPS reductase domain of hFADS2 revealed a co-ordinated network among the Cys residues in this domain. In this model, C312 and C303 are very close to the flavin substrate, consistent with a significantly lowered FAD synthesis rate in C303A and C312A mutants. FAD synthesis is also inhibited by thiol-blocking reagents, suggesting the involvement of free cysteines in the hFADS2 catalytic cycle. Mass spectrometry measurements and titration with thiol reagents on wt hFADS2 and on several individual cysteine/alanine mutants allowed us to detect two stably reduced cysteines (C139 and C241, one for each protein domain), two stable disulfide bridges (C399-C402, C303-C312, both in the PAPS domain), and two unstable disulfides (C39-C50; C440-C464). Whereas the C39-C50 unstable disulfide is located in the MPTb domain and appears to have no catalytic relevance, a cysteine-based redox switch may involve formation and breakdown of a disulfide between C440 and C464 in the PAPS domain.

  13. Overexpression of PIP2;5 aquaporin alleviates effects of low root temperature on cell hydraulic conductivity and growth in Arabidopsis.

    Science.gov (United States)

    Lee, Seong Hee; Chung, Gap Chae; Jang, Ji Young; Ahn, Sung Ju; Zwiazek, Janusz J

    2012-05-01

    The effects of low root temperature on growth and root cell water transport were compared between wild-type Arabidopsis (Arabidopsis thaliana) and plants overexpressing plasma membrane intrinsic protein 1;4 (PIP1;4) and PIP2;5. Descending root temperature from 25°C to 10°C quickly reduced cell hydraulic conductivity (L(p)) in wild-type plants but did not affect L(p) in plants overexpressing PIP1;4 and PIP2;5. Similarly, when the roots of wild-type plants were exposed to 10°C for 1 d, L(p) was lower compared with 25°C. However, there was no effect of low root temperature on L(p) in PIP1;4- and PIP2;5-overexpressing plants after 1 d of treatment. When the roots were exposed to 10°C for 5 d, L(p) was reduced in wild-type plants and in plants overexpressing PIP1;4, whereas there was still no effect in PIP2;5-overexpressing plants. These results suggest that the gating mechanism in PIP1;4 may be more sensitive to prolonged low temperature compared with PIP2;5. The reduction of L(p) at 10°C in roots of wild-type plants was partly restored to the preexposure level by 5 mm Ca(NO(3))(2) and protein phosphatase inhibitors (75 nm okadaic acid or 1 μm Na(3)VO(4)), suggesting that aquaporin phosphorylation/dephosphorylation processes were involved in this response. The temperature sensitivity of cell water transport in roots was reflected by a reduction in shoot and root growth rates in the wild-type and PIP1;4-overexpressing plants exposed to 10°C root temperature for 5 d. However, low root temperature had no effect on growth in plants overexpressing PIP2;5. These results provide strong evidence for a link between growth at low root temperature and aquaporin-mediated root water transport in Arabidopsis.

  14. Isoform expression in the multiple soluble malate dehydrogenase of Hoplias malabaricus (Erythrinidae, Characiformes

    Directory of Open Access Journals (Sweden)

    M. R. Aquino-Silva

    Full Text Available Kinetic properties and thermal stabilities of Hoplias malabaricus liver and skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to further study the possible sMDH-A* locus duplication evolved from a recent tandem duplication. Both A (A1 and A2 and B isoforms had similar optima pH (7.5-8.0. While Hoplias A isoform could not be characterized as thermostable, B could as thermolabile. A isoforms differed from B isoform in having higher Km values for oxaloacetate. The possibly duplicated A2 isoform showed higher substrate affinity than the A1. Hoplias duplicated A isoforms may influence the direction of carbon flow between glycolisis and gluconeogenesis.

  15. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

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    Ralph D Hector

    Full Text Available Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5 cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR, which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  16. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Science.gov (United States)

    Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  17. Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells.

    Science.gov (United States)

    Bulgakova, Natalia A; Rentsch, Michaela; Knust, Elisabeth

    2010-11-15

    Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis.

  18. Differential expression of syndecan isoforms during mouse incisor amelogenesis.

    Science.gov (United States)

    Muto, Taro; Miyoshi, Keiko; Munesue, Seiichi; Nakada, Hiroshi; Okayama, Minoru; Matsuo, Takashi; Noma, Takafumi

    2007-08-01

    Syndecans are transmembranous heparan sulfate proteoglycans (HSPGs) with covalently attached glycosaminoglycan side-chains located on the cell surface. The mammalian syndecan family is composed of four types of syndecans (syndecan-1 to -4). Syndecans interact with the intracellular cytoskeleton through the cytoplasmic domains of their core proteins and membrane proteins, extracellular enzymes, growth factors, and matrix components, through their heparan-sulfate chains, to regulate developmental processes.Here, as a first step to assess the possible roles of syndecan proteins in amelogenesis, we examined the expression patterns of all syndecan isoforms in continuously growing mouse incisors, in which we can overview major differentiation stages of amelogenesis at a glance. Understanding the expression domain of each syndecan isoform during specific developmental stages seems useful for investigating their physiological roles in amelogenesis. Immunohistochemical analysis of syndecan core proteins in the lower incisors from postnatal day 1 mice revealed spatially and temporally specific expression patterns, with syndecan-1 expressed in undifferentiated epithelial and mesenchymal cells, and syndecan-2, -3, and -4 in more differentiated cells. These findings suggest that each syndecan isoform functions distinctly during the amelogenesis of the incisors of mice.

  19. Aquaporins-2 and -4 regulate glycogen metabolism and survival during hyposmotic-anoxic stress in Caenorhabditis elegans.

    Science.gov (United States)

    LaMacchia, John C; Roth, Mark B

    2015-07-15

    Periods of oxygen deprivation can lead to ion and water imbalances in affected tissues that manifest as swelling (edema). Although oxygen deprivation-induced edema is a major contributor to injury in clinical ischemic diseases such as heart attack and stroke, the pathophysiology of this process is incompletely understood. In the present study we investigate the impact of aquaporin-mediated water transport on survival in a Caenorhabditis elegans model of edema formation during complete oxygen deprivation (anoxia). We find that nematodes lacking aquaporin water channels in tissues that interface with the surrounding environment display decreased edema formation and improved survival rates in anoxia. We also find that these animals have significantly reduced demand for glycogen as an energetic substrate during anoxia. Together, our data suggest that reductions in membrane water permeability may be sufficient to induce a hypometabolic state during oxygen deprivation that reduces injury and extends survival limits.

  20. Effect of hyperbaric oxygen preconditioning on the expression of aquaporin 5 in rats with high-altitude pulmonary edema

    Institute of Scientific and Technical Information of China (English)

    LI Zhuo; ZHAO Li-ming; WANG Cong; LIU Xue-hua; GAO Chun-jin

    2012-01-01

    Objective To investigate the possibility of applying hyperbaric oxygen preconditioning(HBO-PC)to reduce or prevent high-altitude pulmonary edema(HAPE)and to detect whether aquaporin 5(AQP5)are involved in HAPE pathogenesis.Methods Thirty eight rats were divided into three groups:the control,the HBO-PC and the HAPE groups.Western blot and real-time PCR were used to analyze the expression of AQP5 in the lungs.The wet-to-dry weight ratio(W/D weight ratio)and morphology of the lung were also examined.Results Lung W/D weight ratio in the HAPE group(4.36±0.53)compared with that of the control group(3.90±0.15)(P<0.05),were marked interstitial edema in all the lung sections in the HAPE group,and fewer changes in 10 of 13 sections in the HBO-PC group.Lung-injury scores of the HBO-PC group were much lower than those of the HAPE group.AQP5 of the HAPE group decreased significantly at the protein and gene levels compared with those in the control group(P<0.01,P<0.05).Protein and mRNA expression of AQP5 in the HBO-PC group were much higher than those of the HAPE group(P<0.05,P<0.01).Lung injuries in the HAPE rats were related positively to the AQP5 expression in the lung(protein expression r=-0.635,P<0.01;mRNA expression r=-0.399,P<0.05).Conclusions We concluded that HBO-PC could alleviate lung injury in rats caused by high-altitude hypobaric hypoxia and reduce HAPE incidence.HAPE in rats was associated with the down-regulation of expression of AQP5 in the lungs.This down-egulation could be attenuated by HBO-PC.This study is the first one introducing HBO-PC in the prevention of HAPE,and the first investigation on the expression of AQP5 in a rat model of HAPE.

  1. Aquaporin 0 plays a pivotal role in refractive index gradient development in mammalian eye lens to prevent spherical aberration

    Energy Technology Data Exchange (ETDEWEB)

    Kumari, S. Sindhu [Physiology and Biophysics, Stony Brook University, Stony Brook, NY (United States); Varadaraj, Kulandaiappan, E-mail: kulandaiappan.varadaraj@stonybrook.edu [Physiology and Biophysics, Stony Brook University, Stony Brook, NY (United States); SUNY Eye Institute, New York, NY (United States)

    2014-10-03

    Highlights: • Intact AQP0 functions as fiber cell-to-fiber cell adhesion protein. • AQP0 facilitates reduction in extracellular space and lens water content. • AQP0 adhesion function aids in lens refractive index gradient (RING) formation. • AQP0 prevents lens spherical aberration by establishing RING. • AQP0 is critical for lens transparency and homeostasis. - Abstract: Aquaporin 0 (AQP0) is a transmembrane channel that constitutes ∼45% of the total membrane protein of the fiber cells in mammalian lens. It is critical for lens transparency and homeostasis as mutations and knockout cause autosomal dominant lens cataract. AQP0 functions as a water channel and as a cell-to-cell adhesion (CTCA) molecule in the lens. Our recent in vitro studies showed that the CTCA function of AQP0 could be crucial to establish lens refractive index gradient (RING). However, there is a lack of in vivo data to corroborate the role of AQP0 as a fiber CTCA molecule which is critical for creating lens RING. The present investigation is undertaken to gather in vivo evidence for the involvement of AQP0 in developing lens RING. Lenses of wild type (WT) mouse, AQP0 knockout (heterozygous, AQP0{sup +/−}) and AQP0 knockout lens transgenically expressing AQP1 (heterozygous AQP0{sup +/−}/AQP1{sup +/−}) mouse models were used for the study. Data on AQP0 protein profile of intact and N- and/or C-terminal cleaved AQP0 in the lens by MALDI-TOF mass spectrometry and SDS–PAGE revealed that outer cortex fiber cells have only intact AQP0 of ∼28 kDa, inner cortical and outer nuclear fiber cells have both intact and cleaved forms, and inner nuclear fiber cells have only cleaved forms (∼26–24 kDa). Knocking out of 50% of AQP0 protein caused light scattering, spherical aberration (SA) and cataract. Restoring the lost fiber cell membrane water permeability (P{sub f}) by transgene AQP1 did not reinstate complete lens transparency and the mouse lenses showed light scattering and SA

  2. Aquaporin 0 plays a pivotal role in refractive index gradient development in mammalian eye lens to prevent spherical aberration.

    Science.gov (United States)

    Kumari, S Sindhu; Varadaraj, Kulandaiappan

    2014-10-03

    Aquaporin 0 (AQP0) is a transmembrane channel that constitutes ∼45% of the total membrane protein of the fiber cells in mammalian lens. It is critical for lens transparency and homeostasis as mutations and knockout cause autosomal dominant lens cataract. AQP0 functions as a water channel and as a cell-to-cell adhesion (CTCA) molecule in the lens. Our recent in vitro studies showed that the CTCA function of AQP0 could be crucial to establish lens refractive index gradient (RING). However, there is a lack of in vivo data to corroborate the role of AQP0 as a fiber CTCA molecule which is critical for creating lens RING. The present investigation is undertaken to gather in vivo evidence for the involvement of AQP0 in developing lens RING. Lenses of wild type (WT) mouse, AQP0 knockout (heterozygous, AQP0(+/-)) and AQP0 knockout lens transgenically expressing AQP1 (heterozygous AQP0(+/)(-)/AQP1(+/)(-)) mouse models were used for the study. Data on AQP0 protein profile of intact and N- and/or C-terminal cleaved AQP0 in the lens by MALDI-TOF mass spectrometry and SDS-PAGE revealed that outer cortex fiber cells have only intact AQP0 of ∼28kDa, inner cortical and outer nuclear fiber cells have both intact and cleaved forms, and inner nuclear fiber cells have only cleaved forms (∼26-24kDa). Knocking out of 50% of AQP0 protein caused light scattering, spherical aberration (SA) and cataract. Restoring the lost fiber cell membrane water permeability (Pf) by transgene AQP1 did not reinstate complete lens transparency and the mouse lenses showed light scattering and SA. Transmission and scanning electron micrographs of lenses of both mouse models showed increased extracellular space between fiber cells. Water content determination study showed increase in water in the lenses of these mouse models. In summary, lens transparency, CTCA and compact packing of fiber cells were affected due to the loss of 50% AQP0 leading to larger extracellular space, more water content and SA

  3. Parental Involvement

    OpenAIRE

    Ezra S Simon

    2008-01-01

    This study was conducted in Ghana to investigate, (1) factors that predict parental involvement, (2) the relationship between parental home and school involvement and the educational achievement of adolescents, (3) the relationship between parental authoritativeness and the educational achievement of adolescent students, (4) parental involvement serving as a mediator between their authoritativeness and the educational achievement of the students, and (5) whether parental involvement decreases...

  4. Novel treatment for lithium-induced nephrogenic diabetes insipidus rat model using the Sendai-virus vector carrying aquaporin 2 gene.

    Science.gov (United States)

    Suga, Hidetaka; Nagasaki, Hiroshi; Kondo, Taka-Aki; Okajima, Yoshiki; Suzuki, Chizuko; Ozaki, Nobuaki; Arima, Hiroshi; Yamamoto, Tokunori; Ozaki, Noriyuki; Akai, Masaro; Sato, Aiko; Uozumi, Nobuyuki; Inoue, Makoto; Hasegawa, Mamoru; Oiso, Yutaka

    2008-11-01

    Congenital nephrogenic diabetes insipidus (NDI) is a chronic disorder involving polyuria and polydipsia that results from unresponsiveness of the renal collecting ducts to the antidiuretic hormone vasopressin. Either of the genetic defects in vasopressin V2 receptor or the water channel aquaporin 2 (AQP2) cause the disease, which interfere the water reabsorption at the epithelium of the collecting duct. An unconscious state including a perioperative situation can be life threatening because of the difficulty to regulate their water balance. The Sendai virus (SeV) vector system deleting fusion protein (F) gene (SeV/DeltaF) is considered most suitable because of the short replication cycle and nontransmissible character. An animal model for NDI with reduced AQP2 by lithium chloride was used to develop the therapy. When the SeV/DeltaF vector carrying a human AQP2 gene (AQP2-SeV/DeltaF) was administered retrogradely via ureter to renal pelvis, AQP2 was expressed in the renal collecting duct to reduce urine output and water intake by up to 40%. In combination with the retorograde administration to pelvis, this system could be the cornerstone for the applicable therapies on not only NDI patients but also other diseases associate with the medullary collecting duct.

  5. The subcellular distribution of aquaporin 5 in the cochlea reveals a water shunt at the perilymph-endolymph barrier.

    Science.gov (United States)

    Hirt, B; Penkova, Z H; Eckhard, A; Liu, W; Rask-Andersen, H; Müller, M; Löwenheim, H

    2010-07-28

    Aquaporins are membrane water channel proteins that have also been identified in the cochlea. Auditory function critically depends on the homeostasis of the cochlear fluids perilymph and endolymph. In particular, the ion and water regulation of the endolymph is essential for sensory transduction. Within the cochlear duct the lateral wall epithelium has been proposed to secrete endolymph by an aquaporin-mediated flow of water across its epithelial tight junction barrier. This study identifies interspecies differences in the cellular distribution of aquaporin 5 (AQP5) in the cochlear lateral wall of mice, rats, gerbils and guinea pigs. In addition the cellular expression pattern of AQP5 is described in the human cochlea. Developmental changes in rats demonstrate longitudinal and radial gradients along the cochlear duct. During early postnatal development a pancochlear expression is detected. However a regression to the apical quadrant and limitation to outer sulcus cells (OSCs) is observed in the adult. This developmental loss of AQP5 expression in the basal cochlear segments coincides with a morphological loss of contact between OSCs and the endolymph. At the subcellular level, AQP5 exhibits polarized expression in the apical plasma membrane of the OSCs. Complementary, the basolateral membrane in the root processes of the OSCs exhibits AQP4 expression. This differential localization of AQP5 and AQP4 in the apical and basolateral membranes of the same epithelial cell type suggests a direct aquaporin-mediated transcellular water shunt between the perilymph and endolymph in the OSCs of the cochlear lateral wall. In the human cochlea these findings may have pathophysiological implications attributed to a dysfunctional water regulation by AQP5 such as endolymphatic hydrops (i.e. in Meniere's disease) or sensorineural hearing loss (i.e. in Sjögren's syndrome).

  6. Genome-Wide Identification and Expression Analyses of Aquaporin Gene Family during Development and Abiotic Stress in Banana

    OpenAIRE

    Hu, Wei; Hou, Xiaowan; Huang, Chao; Yan, Yan; Tie, Weiwei; Ding, Zehong; Wei, Yunxie; Liu, Juhua; Miao, Hongxia; Lu, Zhiwei; li, Meiying; Xu, Biyu; Jin, Zhiqiang

    2015-01-01

    Aquaporins (AQPs) function to selectively control the flow of water and other small molecules through biological membranes, playing crucial roles in various biological processes. However, little information is available on the AQP gene family in bananas. In this study, we identified 47 banana AQP genes based on the banana genome sequence. Evolutionary analysis of AQPs from banana, Arabidopsis, poplar, and rice indicated that banana AQPs (MaAQPs) were clustered into four subfamilies. Conserved...

  7. Molecular characterization and expression profiles of four transformer-2 isoforms in the Chinese mitten crab Eriocheir sinensis

    Science.gov (United States)

    Luo, Danli; Liu, Yuan; Hui, Min; Song, Chengwen; Liu, Hourong; Cui, Zhaoxia

    2016-09-01

    The transformer-2 (tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster. In this study, sequences and expression profiles of tra-2 in the Chinese mitten crab Eriocheir sinensis were characterized. Four tra-2 isoforms, designated as Estra-2a, Estra-2b, Estra-2c and Estra-2d, were isolated. They all contained an RNA-recognition motif (RRM) and a linker region, which shared high similarity with other reported tra-2s. Sequence analysis revealed that Estra-2a, Estra-2b and Estra-2c are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA. Compared with the other three isoforms, Estra-2d lacks the RS2 domain. Quantitative real-time PCR showed that all four isoforms were highly expressed in the fertilized egg, and in the 2-4 cell and blastula stages compared with larval stages (P≤ 0.01), suggesting their maternal origin in early embryonic developmental stages. Notably, Estra-2a was highly expressed in male somatic tissues, while Estra-2c was significantly highly expressed in the ovary. These results suggest that Estra-2c is involved in sexual differentiation of the Chinese mitten crab. Our findings provide basic information for further functional studies of the tra-2 gene/protein in this species.

  8. RNA-Seq analysis of the parietal cortex in Alzheimer's disease reveals alternatively spliced isoforms related to lipid metabolism.

    Science.gov (United States)

    Mills, James D; Nalpathamkalam, Thomas; Jacobs, Heidi I L; Janitz, Caroline; Merico, Daniele; Hu, Pingzhao; Janitz, Michael

    2013-03-01

    The parietal cortex of the human brain plays a unique role in the coordination of movement and in the integration of signals from the other cortices. Because of its extensive connections and involvement in many higher-order cognitive functions, neurodegenerative changes in the parietal lobe are believed to be crucial in the early symptoms of Alzheimer's disease (AD). Little is known about the transcriptome of this part of the human brain or how it is perturbed by the neurodegenerative process. To that end, we performed mRNA sequencing using the Illumina RNA-Seq technique on samples derived from normal and AD parietal lobes. Gene expression analysis evaluating alternatively spliced isoform expression and promoter usage revealed surprisingly elevated transcriptome activity in the AD condition. This phenomenon was particularly apparent in the alternative usage of transcriptional start sites. A Gene Ontology analysis of the differentially expressed genes revealed enrichment in the functional pathways related to lipid metabolism, thus highlighting the importance of astrocyte activity in the neurodegenerative process. We also identified an upregulation of the diazepam-binding inhibitor (DBI) gene in AD, as the result of a splicing switch toward shorter, intron-retaining isoforms driven by alternative promoters and was coupled with a simultaneous decrease in the abundance of protein-coding transcripts. These two DBI isoforms have not been described previously.

  9. Molecular identity, expression and functional analysis of interleukin-lα and its isoforms in rat testis

    Institute of Scientific and Technical Information of China (English)

    T.Sultana; K.V.Svechnikov; K.Gustafsson; A.Wahlgren; E.Tham; G.Weber; O.Soder

    2004-01-01

    Interleukin-1α(IL-1α) is a proinflammatory cytokine that has also been found to act as a paracrine mediator involved in the regulation of testicular functions. The present review provides an overview of the role of IL-1α in testicular physiology. Bioactive IL-1α isolated from adult rat testis was found to consist of three distinct immunoreactive protein species with apparent sizes of 45, 24 and 19 kDa. These isoforms showed bioactivity in athymocyte proliferation and steroidogenesis assays with different biopotencies. The background of the molecular heterogeneity and processing, secretion and regulation of the isoforms of testicular IL-1α are discussed. All three isoforms have been found to be secreted into the testis tubular lumen and interstitial space. We have provided evidence that IL- 1α is a paracrine factor that may be of importance in, e.g., the regulation of Leydig cell steroidogenesis.Pathophysiologically, testicular IL-1α may contribute to testicular relapse of acute lymphocytic leukemia in boys.

  10. Two isoforms of glutamate decarboxylase in Arabidopsis are regulated by calcium/calmodulin and differ in organ distribution.

    Science.gov (United States)

    Zik, M; Arazi, T; Snedden, W A; Fromm, H

    1998-08-01

    The nucleotide sequences of cDNAs encoding two isoforms of Arabidopsis glutamate decarboxylase, designated GAD1 (57.1 kDa) and GAD2 (56.1 kDa) and sharing 82% identical amino acid sequences, were determined. The recombinant proteins bound [35S] calmodulin (CaM) in the presence of calcium, and a region of 30-32 amino acids from the C-terminal of each isoform was sufficient for CaM binding when fused to glutathione S-transferase. Full-length GAD1 and GAD2 were expressed in Sf9 insect cells infected with recombinant baculovirus vectors. Recombinant proteins were partially purified by CaM affinity chromatography and were found to exhibit glutamate decarboxylase activity, which was dependent on the presence of Ca2+/CaM at pH 7.3. Southern hybridizations with GAD gene-specific probes suggest that Arabidopsis possesses one gene related to GAD1 and one to GAD2. Northern hybridization and western blot analysis revealed that GAD1 was expressed only in roots and GAD2 in roots, leaves, inflorescence stems and flowers. Our study provides the first evidence for the occurrence of multiple functional Ca2+/CaM-regulated GAD gene products in a single plant, suggesting that regulation of Arabidopsis GAD activity involves modulation of isoform-specific gene expression and stimulation of the catalytic activity of GAD by calcium signalling via CaM.

  11. Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

    Directory of Open Access Journals (Sweden)

    Insa Bultmann-Mellin

    2015-04-01

    Full Text Available Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C, which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S−/−, the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S−/− and Ltbp4-null (Ltbp4−/− mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM. Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C.

  12. Functional Comparison of 45 Naturally Occurring Isoforms of the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT).

    Science.gov (United States)

    Callaghan, Paul S; Hassett, Matthew R; Roepe, Paul D

    2015-08-18

    At least 53 distinct isoforms of Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein are expressed in strains or isolates of P. falciparum malarial parasites from around the globe. These parasites exhibit a range of sensitivities to chloroquine (CQ) and other drugs. Mutant PfCRT is believed to confer cytostatic CQ resistance (CQR(CS)) by transporting CQ away from its DV target (free heme released upon hemoglobin digestion). One theory is that variable CQ transport catalyzed by these different PfCRT isoforms is responsible for the range of CQ sensitivities now found for P. falciparum. Alternatively, additional mutations in drug-selected parasites, or additional functions of PfCRT, might complement PfCRT-mediated CQ transport in conferring the range of observed resistance phenotypes. To distinguish between these possibilities, we recently optimized a convenient method for measuring PfCRT-mediated CQ transport, involving heterologous expression in Saccharomyces cerevisiae. Here, we use this method to quantify drug transport activity for 45 of 53 of the naturally occurring PfCRT isoforms. Data show that variable levels of CQR likely depend upon either additional PfCRT functions or additional genetic events, including perhaps changes that influence DV membrane potential. The data also suggest that the common K76T PfCRT mutation that is often used to distinguish a P. falciparum CQR phenotype is not, in and of itself, a fully reliable indicator of CQR status.

  13. Changes in aquaporin gene expression and magnetic resonance imaging of water status in peach tree flower buds during dormancy.

    Science.gov (United States)

    Yooyongwech, Suravoot; Horigane, Akemi K; Yoshida, Mitsuru; Yamaguchi, Masami; Sekozawa, Yoshihiro; Sugaya, Sumiko; Gemma, Hiroshi

    2008-11-01

    The movement of cellular water accompanies changes in growth within dormant buds. To further understand this process, accumulation of tonoplast deltaTIP1 and plasma membrane PIP2 aquaporin transcripts was measured by quantitative reverse transcriptase-polymerase chain reaction and the water dynamics in dormant peach (Prunus persica L.) flower buds was studied by magnetic resonance imaging. Proton density (PD), spin-spin relaxation time (T(2)) and apparent diffusion coefficient (ADC) were used to observe water dynamics during dormancy. The expression of deltaTIP1 and PIP2 aquaporins, PD and T(2) in the upper part of the bud including primordia, in the basal part of the bud and the bud trace increased earlier in the low-chill cultivar 'Coral' than in the high-chill cultivar 'Kansuke Hakuto,' reflecting the difference in timing for the end of endodormancy in the two cultivars. deltaTIP1 mRNA accumulated mainly in the basal part of the bud, whereas PIP2 mRNA was detected mainly in the upper part. These findings may reflect the activation of inter- and intracell communication through membrane transport properties of aquaporins resulting in a gradual increase in water content to that required for bud activity at the end of endodormancy. An apparent decrease in the expression of deltaTIP1 and PIP2 mRNAs was, however, observed in late winter in some portions of the buds of both cultivars just before sprouting.

  14. The natural profilin from Russian thistle (Salsola kali) contains a low IgE-binding ability isoform--molecular and immunological characterization.

    Science.gov (United States)

    Mas, Salvador; Barderas, Rodrigo; Colás, Carlos; Quiralte, Joaquín; Rodríguez, Rosalía; Villalba, Mayte

    2012-12-01

    Chenopodiaceae pollens such as those from Salsola kali and Chenopodium album are important causes of allergy in Mediterranean areas because of the progress of desertification in European countries. Among the various allergenic protein families, profilins constitute a group of pan-allergens that are involved in polysensitization and pollen-food allergy syndrome. Two-dimensional electrophoresis analysis of S. kali profilin highlighted a polymorphic pattern, with several isoforms that have different molecular features (isoelectric point and molecular mass) and immunological features. Two isoforms have been cloned and sequenced. Sal k 4.02 and Sal k 4.03 displayed non-conservative amino acid changes in critical positions of the IgE epitopes. Both isoforms were produced in Escherichia coli and structurally and spectroscopically characterized. Changes in the electrophoretic mobility and in their IgG and IgE immunological behavior were observed in comparison with Che a 2, their counterpart from C. album. The IgE-binding ability of Sal k 4.03 is similar to that of Che a 2, whereas Sal k 4.02 showed a 35% reduction in IgE binding in 86% of patients, suggesting a hypoallergenic character. Three-dimensional modeling allowed us to propose which amino acid residues are involved in those immunological changes based on epitope mapping studies previously performed in other profilins. These profilin isoforms constitute suitable candidates for specific immunotherapy with recombinant allergens.

  15. Role of specific cytochrome P450 isoforms in the conversion of phenoxypropoxybiguanide analogs in human liver microsomes to potent antimalarial dihydrotriazines.

    Science.gov (United States)

    Diaz, Damaris S; Kozar, Michael P; Smith, Kirsten S; Asher, Constance O; Sousa, Jason C; Schiehser, Guy A; Jacobus, David P; Milhous, Wilbur K; Skillman, Donald R; Shearer, Todd W

    2008-02-01

    Phenoxypropoxybiguanides, such as PS-15, are antimalarial prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, WR99210, the active metabolite of PS-15, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Recently, in vitro metabolism of a new series of phenoxypropoxybiguanide analogs has examined the production of the active triazine metabolites by human liver microsomes. The purpose of this investigation was to elucidate the primary cytochrome P450 isoforms involved in the production of active metabolites in the current lead candidate. By using expressed human recombinant isoform preparations, specific chemical inhibitors, and isoform-specific inhibitory antibodies, the primary cytochrome P450 isoforms involved in the in vitro metabolic activation of JPC-2056 were elucidated. Unlike proguanil, which is metabolized primarily by CYP2C19, the results indicate that CYP3A4 plays a more important role in the metabolism of both PS-15 and JPC-2056. Whereas CYP2D6 appears to play a major role in the metabolism of PS-15 to WR99210, it appears less important in the conversion of JPC-2056 to JPC-2067. These results are encouraging, considering the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, because the current clinical prodrug candidate from this series may be less dependent on these enzymes for metabolic activation.

  16. Effects of exogenous 5-aminolevulinic acid on PIP1 and NIP aquaporin gene expression in seedlings of cucumber cultivars subjected to salinity stress.

    Science.gov (United States)

    Yan, F; Qu, D; Zhao, Y Y; Hu, X H; Zhao, Z Y; Zhang, Y; Zou, Z R

    2014-01-22

    Aquaporins play a direct role in plant water relation under salt stress, but the effects of 5-aminolevulinic acid (ALA) on aquaporin gene expression in salt-treated plants remain unknown. This study investigated the potential effects of exogenous ALA (50 mg/dm3) on aquaporin expression levels under salt stress (75 mM NaCl) in the salt-sensitive (Jinchun No.4) and the relatively salt-tolerant cucumber (Jinyou No.1) seedlings. The expressions of cucumber PIP aquaporin gene (CsPIP1:1) and cucumber NIP aquaporin gene (CsNIP) were analyzed in 20-day-old seedling leaves at 2, 4, 8, 16, and 24 h after ALA treatment. After treatment with saline alone and ALA alone, CsPIP1:1 and CsNIP gene expression levels in the 2 cucumber cultivars increased to maximum at 2 h. The aquaporin gene expression in salt-treated cucumber seedling leaves was considerably higher than that in leaves subjected to exogenous ALA. Further, the aquaporin expression levels in Jinchun No.4 were higher than those in Jinyou No.1, reaching 5.20- and 2-fold induction levels, respectively. After treatment with both ALA and NaCl, the CsNIP gene expression was downregulated in both the cucumber cultivars, while that of CsPIP1:1 decreased at 2 h and then increased to 3.8-fold in Jinchun No.4. In Jinyou No.1, CsPIP1:1 gene expression gradually increased to 2.3-fold at 4 h, followed by a decline in expression. The results indicated that ALA might delay and counteract the upregulated expression of CsPIP1:1 and CsNIP genes in cucumber seedlings under NaCl stress. Thus, salt tolerance of cucumber seedlings might be enhanced by ALA application.

  17. Novel intracellular N-terminal truncated matrix metalloproteinase-2 isoform in skeletal muscle ischemia-reperfusion injury.

    Science.gov (United States)

    Joshi, Sunil K; Lee, Lawrence; Lovett, David H; Kang, Heejae; Kim, Hubert T; Delgado, Cynthia; Liu, Xuhui

    2016-03-01

    Ischemia-reperfusion injury (IRI) occurs when blood returns to tissues following a period of ischemia. Reintroduction of blood flow results in the production of free radicals and reactive oxygen species that damage cells. Skeletal muscle IRI is commonly seen in orthopedic trauma patients. Experimental studies in other organ systems have elucidated the importance of extracellular and intracellular matrix metalloproteinase-2 (MMP-2) isoforms in regulating tissue damage in the setting of oxidant stress resulting from IRI. Although the extracellular full-length isoform of MMP-2 (FL-MMP-2) has been previously studied in the setting of skeletal muscle IRI, studies investigating the role of the N-terminal truncated isoform (NTT-MMP-2) in this setting are lacking. In this study, we first demonstrated significant increases in FL- and NTT-MMP-2 gene expression in C2C12 myoblast cells responding to re-oxygenation following hypoxia in vitro. We then evaluated the expression of FL- and NTT-MMP-2 in modulating skeletal muscle IRI using a previously validated murine model. NTT-MMP-2, but not FL-MMP-2 expression was significantly increased in skeletal muscle following IRI. Moreover, the expression of toll-like receptors (TLRs) -2 and -4, IL-6, OAS-1A, and CXCL1 was also significantly up-regulated following IRI. Treatment with the potent anti-oxidant pyrrolidine dithiocarbamate (PDTC) significantly suppressed NTT-MMP-2, but not FL-MMP-2 expression and improved muscle viability following IRI. This data suggests that NTT-MMP-2, but not FL-MMP-2, is the major isoform of MMP-2 involved in skeletal muscle IRI.

  18. Inhibition of carbonic anhydrase isoforms I, II, IX and XII with secondary sulfonamides incorporating benzothiazole scaffolds.

    Science.gov (United States)

    Petrou, Anthi; Geronikaki, Athina; Terzi, Emine; Guler, Ozen Ozensoy; Tuccinardi, Tiziano; Supuran, Claudiu T

    2016-12-01

    Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the fundamental reaction of CO2 hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. A series of benzothiazole-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I and hCA II and the transmembrane hCA IX and hCA XII. Several of the investigated derivatives showed interesting inhibition activity and selectivities for inhibiting hCA IX and hCA XII over the off-target ones hCA I and hCA II. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.

  19. ROCK in CNS: Different Roles of Isoforms and Therapeutic Target for Neurodegenerative Disorders.

    Science.gov (United States)

    Chong, Cheong-Meng; Ai, Nana; Lee, Simon Ming-Yuen

    2017-01-01

    Rho-associated protein kinase (ROCK) is a serine-threonine kinase originally identified as a crucial regulator of actin cytoskeleton. Recent studies have defined new functions of ROCK as a critical component of diverse signaling pathways in neurons. In addition, inhibition of ROCK causes several biological events such as increase of neurite outgrowth, axonal regeneration, and activation of prosurvival Akt. Thus, it has attracted scientist's strong attentions and considered ROCK as a promising therapeutic target for the treatment of neurodegenerative disorders including Alzheimer disease, Parkinson's disease, Huntington';s disease, multiple sclerosis, and amyotrophic lateral sclerosis. However, ROCK has two highly homologous isoforms, ROCK1 and ROCK2. Accumulated evidences indicate that ROCK1 and ROCK2 might involve in distinct cellular functions in central nervous system (CNS) and neurodegenerative processes. This review summarizes recent updates regarding ROCK isoformspecific functions in CNS and the progress of ROCK inhibitors in preclinical studies for neurodegenerative diseases.

  20. In vivo generation of neurotoxic prion protein: role for hsp70 in accumulation of misfolded isoforms.

    Directory of Open Access Journals (Sweden)

    Pedro Fernandez-Funez

    2009-06-01

    Full Text Available Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP(C converts into a misfolded isoform (PrP(Sc with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP(C; in older flies, PrP misfolds, acquires biochemical and structural properties of PrP(Sc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP(Sc-specific conformational epitopes. In contrast to PrP(Sc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP(Sc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP(Sc-like conformers and protects against PrP-dependent