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Channel Protein (channel + protein)
Kinds of Channel Protein Selected AbstractsPituitary and autonomic responses to cold exposures in manACTA PHYSIOLOGICA, Issue 4 2005J. Leppäluoto Abstract This review presents hormonal responses to various cold exposures and their calorigenic effects in man and some animals. Previous studies in rats have shown that cold exposures activate the hypothalamic-pituitary-thyroid axis. Increased thyroid hormone concentrations lead to heat production via general stimulation of metabolism (obligatory thermogenesis) and possibly via activation of thyroid hormone receptors and uncoupling protein 1 (UCP 1) and deiodinase enzyme genes in the brown adipose tissue (BAT). In human subjects long-term cold exposures do not seem to activate the pituitary-thyroid axis, but rather accelerate the elimination of triiodothyronine (T3), leading to low serum concentrations of free T3 hormone. In corollary to this a hypothyreotic condition with increased serum thyroid-stimulating hormone and impaired mood and cognitive performance can be observed after long-term cold exposures such as wintering. During cold exposures the sympathetic nerve system is activated and noradrenaline is released to blood circulation and to BAT, where it leads to production of cAMP, lipolysis and free fatty acids. Free fatty acids open the mitochondrial proton channel protein in BAT. Protons enter the mitochondria and inhibit ATP synthesis (uncoupling). By this way energy is transformed into heat (facultatory or adaptive thermogenesis). In adult human subjects the amount of BAT is small and adaptive thermogenesis (non-shivering thermogenesis) has a smaller role. UCP 1 with other uncoupling proteins may have other functions in the control of body weight, sugar balance and formation of reactive oxygen species. [source] Effects of agrin on the expression and distribution of the water channel protein aquaporin-4 and volume regulation in cultured astrocytesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2007Susan Noell Abstract Agrin is a heparan sulfate proteoglycan of the extracellular matrix and is known for organizing the postsynaptic differentiation of the neuromuscular junction. Increasing evidence also suggests roles for agrin in the developing CNS, including the formation and maintenance of the blood,brain barrier. Here we describe effects of agrin on the expression and distribution of the water channel protein aquaporin-4 (AQP4) and on the swelling capacity of cultured astrocytes of newborn mice. If astrocytes were cultured on a substrate containing poly dl -ornithine, anti-AQP4 immunoreactivity was evenly and diffusely distributed. If, however, astrocytes were cultured in the presence of agrin-conditioned medium, we observed an increase in the intensity of AQP4-specific membrane-associated staining. Freeze-fracture studies revealed a clustering of orthogonal arrays of particles, representing a structural equivalent of AQP4, when exogenous agrin was present in the astrocyte cultures. Neuronal and non-neuronal agrin isoforms (agrin A0B0 and agrin A4B8, respectively) were able to induce membrane-associated AQP4 staining. Water transport capacity as well as the density of orthogonal arrays of intramembranous particles was increased in astrocytes cultured with the neuronal agrin isoform A4B8, but not with the endothelial and meningeal isoform A0B0. RT-PCR demonstrated that agrin A4B8 increased the level of the M23 splice variant of AQP4 and decreased the level of the M1 splice variant of AQP4. Implications for the regulation and maintenance of the blood,brain barrier including oedema formation under pathological conditions are discussed. [source] Chronic interleukin-6 alters the level of synaptic proteins in hippocampus in culture and in vivoEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2007Elly J. F. Vereyken Abstract There is now considerable evidence that the level of expression of the proinflammatory cytokine, interleukin-6 (IL-6), is increased in the central nervous system (CNS) during neuroinflammatory conditions such as occurs in neurological disorders and in disease and injury. However, our understanding of the consequences of increased expression of IL-6 on the CNS is still limited, especially with respect to the developing nervous system, which is known to be particularly vulnerable to environmental factors. To address this issue, we investigated the properties of cultured hippocampal neurons exposed chronically to IL-6 during the main period of morphological and physiological development, which occurs during the first 2 weeks of culture. IL-6 was tested at 500 U/mL, considered to reflect a pathophysiologic concentration. The morphological features of neuronal development in the control and IL-6-treated cultures appeared similar. However, Western blot analysis showed a significant reduction in the level of Group-II metabotropic receptors (mGluR2/3) and L-type Ca2+ channels in the IL-6-treated cultures. A similar reduction in mGluR2/3 and L-type Ca2+ channel protein was observed in transgenic mice that over-express IL-6 in the CNS through astrocyte production starting early in development. Analysis of Ca2+ signals produced by spontaneous synaptic network activity in the hippocampal cultures and effects of a mGluR2/3 agonist and antagonist showed that the reduced levels of mGluR2/3 impact on the functional properties of hippocampal synaptic network activity. These results have important implications relative to the mechanisms responsible for altered CNS function during conditions associated with increased levels of IL-6 in the CNS. [source] Aquaporin 4 changes in rat brain with severe hydrocephalusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006Xiaoyan Mao Abstract Hydrocephalus is characterized by impaired cerebrospinal fluid (CSF) flow with enlargement of the ventricular cavities of the brain and progressive damage to surrounding tissue. Bulk water movement is altered in these brains. We hypothesized that increased expression of aquaporins, which are water-permeable channel proteins, would occur in these brains to facilitate water shifts. We used quantitative (real-time) RT-PCR, Western blotting and immunohistochemistry to evaluate the brain expression of aquaporins (AQP) 1, 4, and 9 mRNA and protein in Sprague,Dawley rats rendered hydrocephalic by injection of kaolin into cistern magna. AQP4 mRNA was significantly up-regulated in parietal cerebrum and hippocampus 4 weeks and 9 months after induction of hydrocephalus (P < 0.05). Although Western blot analysis showed no significant change, there was more intense perivascular AQP4 immunoreactivity in cerebrum of hydrocephalic brains at 3,4 weeks after induction. We did not detect mRNA or protein changes in AQP1 (located in choroid plexus) or AQP9 (located in select neuron populations). Kir4.1, a potassium channel protein linked to water flux, exhibited enhanced immunoreactivity in the cerebral cortex of hydrocephalic rats; the perineuronal distribution was entirely different from that of AQP4. These results suggest that brain AQP4 up-regulation might be a compensatory response to maintain water homeostasis in hydrocephalus. [source] A new class of neurotoxin from wasp venom slows inactivation of sodium currentEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2000Yoshinori Sahara Abstract The effects of ,-pompilidotoxin (,-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that ,-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action of ,-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and ,-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of ,-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that ,-PMTX slowed the Na+ channels inactivation process without changing the peak current,voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that ,-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that ,-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of ,-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein. [source] A role of the C-terminus of aquaporin 4 in its membrane expression in cultured astrocytesGENES TO CELLS, Issue 7 2002Ken-ichi Nakahama Background: Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brains, which is localized in the astrocyte plasma membrane. Membrane targeting of AQP4 is essential to perform its function. The mechanism(s) of membrane targeting is not clear in astrocytes. Results: We investigated the role of the C-terminus of AQP4 (short isoform) in its membrane targeting by an expression study of C-terminal mutants of AQP4 in cultured astrocytes. The deletion of 26 C-terminal residues of AQP4 (AQP4,276,301aa) results in the intracellular localization of the protein. However, smaller deletions than 21 C-terminal residues did not alter its plasma membrane localization. These results suggest that C-terminal residues between Val276 and Ile280 play an important role in the expression of AQP4 in the plasma membrane. However, the plasma membrane localization of the AQP4(A276AAAA280) mutant (alanine substitution of Val276 -Ile280 of AQP4) suggests that another signal for membrane targeting exists in the C-terminus of AQP4. The deletion or point mutations of the PDZ binding motif of the AQP4(A276AAAA280) mutant resulted in the intracellular localization of the proteins. These results suggest that the PDZ binding motif may also be involved in the membrane targeting of AQP4. Conclusions: We found that the C-terminal sequence of AQP4 contains two important signals for membrane expression of AQP4 in cultured astrocytes. One is a hydrophobic domain and the other is a PDZ binding motif that exists in the C-terminus. [source] Modulation of activity of the adipocyte aquaglyceroporin channel by plant extractsINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 1 2007M.-M. Cals-Grierson Synopsis The plasma membrane protein, aquaglyceroporin-7 (AQP7) is exclusively expressed in adipocytes and appears to be a channel for glycerol entry and exit. It is possible that by facilitating the opening of these channels, the loss of intracellular glycerol could be encouraged and thus reduce the size of the lipid reservoir. Human preadipocytes and mouse 3T3-L1 preadipocytes were induced to develop an adipocytic phenotype by culture in a semi-defined medium. After 7 days, the expression of AQP7 message had increased by 37-fold, a level which could be further up-regulated by troglitazone or retinoic acid or down-regulated by insulin. The mature adipocytes also expressed immunoreactive aquaporin (AQP) channel protein as assessed by immunocytochemistry and Western blot. The addition of adrenaline to the culture medium stimulated the release of glycerol (blockable by HgCl2). Plant extracts, with potential anti-cellulite properties, were tested for their effect on glycerol elimination. These included wild yam root (Dioscorea opposita), cocoa bean (Theobroma cacao), horse chestnut tree (Aesculus hippocastanum) seed and bark and tomato (Solanum lycopersicum). Of these, D. opposita appeared to induce a dose-dependent glycerol release. The results show that our assay can help to identify modulators of AQP7 channel expression and activation in adipocytes. Résumé Le canal aquaglyceroporine 7 (AQP7) est la voie principale d'entrée et sortie du glycérol dans les adipocytes. Nous avons émis l'hypothèse que la stimulation de ces canaux pourrait augmenter la perte de glycérol des vésicules de stockage des graisses dans les adipocytes et diminuer ainsi le volume des adipocytes. Des pré-adipocytes humains et des pré-adipocytes murins (lignée 3T3 - L1) ont été mis en culture et induits à développer un phénotype d'adipocyte mature différencié. L'expression de l'ARNm AQP7 est multipliée par 37 dans les cellules matures comparativement aux cellules immatures. Le taux d'expression peut être modulé par l'addition de troglitazone, d'acide rétinoique ou d'insuline. Les deux types de cellules (adipocytes matures humains et murins) possèdent une immunoreactivité spécifique de AQP, visualisée par immunocytochimie et par analyse en « Western blot ». Certains actifs susceptibles de moduler le passage de glycérol (et /ou d'augmenter le nombre des canaux actifs) ont étéévalués. L'épinéphrine à 1 ,M (contrôle positif) entraîne une libération maximale de glycérol. Cette réponse est bloquée par HgCl2 (fermeture des canaux AQP7). Plusieurs extraits végétaux ayant un potentiel anti-cellulite ont étéévalués: un extrait de la racine de l'igname sauvage (D. opposita), un extrait de cacao (T. cacao), des extraits du noyau et de l'écorce de marronnier (A. hippocastanum) et un extrait de tomate (S. lycopersicum; fruit). Parmi eux, D. opposita a provoqué une libération de glycérol de manière dose-dépendente. Ces résultats montrent que ce test peut être utile à la recherche cosmétique, pour identifier de nouveaux modulateurs des canaux AQP7 adipocytaires. [source] Translocation of Proteins into MitochondriaIUBMB LIFE, Issue 6 2001Nicholas J. Hoogenraad Abstract The translocase of the outer mitochondrial membrane (TOM) is composed of receptors, a channel protein, and its modulators that function together to import proteins into mitochondria. Although the import pathway of proteins directed to the mitochondrial matrix has been well characterized, recent studies into the import pathway taken by proteins into the other submitochondrial compartments have broadened our understanding into the way the TOM machinery recognizes, interacts, and translocates proteins. [source] Molecular Diversity of Vasotocin-Dependent Aquaporins Closely Associated with Water Adaptation Strategy in Anuran AmphibiansJOURNAL OF NEUROENDOCRINOLOGY, Issue 5 2010M. Suzuki Anuran amphibians represent the first vertebrates that adapted to terrestrial environments, and are successfully distributed around the world, even to forests and arid deserts. Many adult anurans have specialised osmoregulatory organs, in addition to the kidney (i.e. the ventral pelvic skin to absorb water from the external environments and a urinary bladder that stores water and reabsorbs it in times of need). Aquaporin (AQP), a water channel protein, plays a fundamental role in these water absorption/reabsorption processes. The anuran AQP family consists of at least AQP0-AQP5, AQP7-AQP10 and two anuran-specific types, designated as AQPa1 and AQPa2. For the three osmoregulatory organs, AQP3 is constitutively located in the basolateral membrane of the tight-junctioned epithelial cells, allowing water transport between the cytoplasm of these cells and the neighbouring tissue fluid at all times. On the other hand, AQPs at the apical side of the tight epithelial cells are different among these organs, and are named kidney-type AQP2, ventral pelvic skin-type AQPa2 and urinary bladder-type AQPa2. All of them show translocation from the cytoplasmic pool to the apical plasma membrane in response to arginine vasotocin, thereby regulating water transport independently in each osmoregulatory organ. It was further revealed that, in terrestrial and arboreal anurans, the bladder-type AQPa2 is expressed in the pelvic skin, together with the pelvic skin-type AQPa2, potentially facilitating water absorption from the pelvic skin. By contrast, Xenopus has lost the ability to efficiently produce pelvic skin-type AQPa2 (AQP-x3) because Cys-273 of AQP-x3 and/or Cys-273-coding region of AQPx3 mRNA attenuate gene expression at a post-transcriptional step, presumably leading to the prevention of excessive water influx in this aquatic species. Collectively, the acquisition of two forms of AQPa2 and the diversified regulation of their gene expression appears to provide the necessary mechanisms for the evolutionary adaptation of anurans to a wide variety of ecological environments. [source] Enhanced Aquaporin-4 immunoreactivity in sporadic Creutzfeldt-Jakob diseaseNEUROPATHOLOGY, Issue 4 2007Yasushi Iwasaki Aquaporin-4 (AQP-4) is a water channel protein located on the plasma membrane of astrocytes and is regulated under various conditions. In the present study, a series of brains with sporadic Creutzfeldt-Jakob disease (sCJD) were investigated to determine the possible contribution of AQP-4 in the development of sCJD pathology. Six cases of subacute spongiform encephalopathy (SSE) and four cases of panencephalopathic (PE)-type sCJD were included. Increased AQP-4 immunoreactivity compared to that in controls was observed in all sCJD patients, particularly in the cerebral neocortex and cerebellar cortex. AQP-4 immunoreactivity was present in the cell bodies and processes of protoplasmic astrocytes in SSE and around cell bodies and processes of hypertrophic astrocytes in PE-type sCJD. Analysis of serial sections showed the development of sCJD pathology, particularly in neocortical lesions, as follows: PrP deposition; spongiform change and gliosis; enhanced staining for AQP-4; hypertrophic astrocytosis; and neuronal loss and tissue rarefaction. Strong AQP-4 immunoreactivity was present in burnt-out lesions such as those of status spongiosus. These results indicate that increased AQP-4 expression in sCJD is an early pathologic event and appears to remain until the late disease stage. We suggest that increased expression of AQP-4 is a pathologic feature of sCJD. [source] Knockdown resistance to DDT and pyrethroids: from target-site mutations to molecular modellingPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 11 2008TG Emyr Davies Abstract Naturally derived insecticides such as pyrethrum and man-made insecticides such as DDT and the synthetic pyrethroids act on the voltage-gated sodium channel proteins found in insect nerve-cell membranes. The correct functioning of these channels is essential for the normal transmission of nerve impulses, and this process is disrupted by binding of the insecticides, leading to paralysis and eventual death. Some insect pest populations have evolved modifications of the sodium channel protein that inhibit the binding of the insecticide and result in the insect developing resistance. This perspective outlines the current understanding of the molecular processes underlying target-site resistance to these insecticides (termed kdr and super-kdr), and how this knowledge may in future contribute to the design of novel insecticidal compounds. Copyright © 2008 Society of Chemical Industry [source] Linkage of genes for sodium channel and cytochrome P450 (CYP6B10) in Heliothis virescensPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 2 2002Sujin Park Abstract Genetic linkage of hscp (heliothis sodium channel protein) and CYP6B10 was discovered in Heliothis virescens. The hscp gene encodes the sodium channel target of pyrethroid insecticides and cytochrome P450 genes encode important enzymes involved in detoxication of various pesticides. Previously, two mechanisms, nerve insensitivity due to sodium channel and synergism by propynyl aryl ethers, were observed in pyrethroid-resistant H virescens and were not separated by repeated back-crossing. We hypothesized genetic linkage of target site insensitivity and monooxygenase-mediated detoxication. Single nucleotide polymorphisms were discovered in IIS6 of hscp; Hpy of hscp and CYP6B10. Segregation of these and other markers was tested in backcrosses. We observed co-segregation of hscp to CYP6B10, but both genes assorted independently of y, ye and sex. Genes y and ye assorted independently of each other. This was the first observation of linkage between genes controlling detoxication and sodium ion channel insensitivity in a species known to express high levels of pyrethroid resistance. Linkage was not likely because this species has 31 chromosomes; therefore, we will investigate the possibility of a resistance cassette. We expect similar linkage in other noctuid pests. © 2001 Society of Chemical Industry [source] Effects of Low Power Laser Irradiation on Intracellular Calcium and Histamine Release in RBL-2H3 Mast CellsPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2007Wen-Zhong Yang Although laser irradiation has been reported to promote skin wound healing, the mechanism is still unclear. As mast cells are found to accumulate at the site of skin wounds we hypothesized that mast cells might be involved in the biological effects of laser irradiation. In this work the mast cells, RBL-2H3, were used in vitro to investigate the effects of laser irradiation on cellular responses. After laser irradiation, the amount of intracellular calcium ([Ca2+]i) was increased, followed by histamine release, as measured by confocal fluorescence microscopy with Fluo-3/AM staining and a fluorescence spectrometer with o -phthalaldehyde staining, respectively. The histamine release was mediated by the increment of [Ca2+]i from the influx of the extracellular buffer solution through the cation channel protein, transient receptor potential vanilloid 4 (TRPV4). The TRPV4 inhibitor, Ruthenium Red (RR) can effectively block such histamine release, indicating that TRPV4 was the key factor responding to laser irradiation. These induced responses of mast cells may provide an explanation for the biological effects of laser irradiation on promoting wound healing, as histamine is known to have multi-functions on accelerating wound healing. [source] Cytoskeleton-associated, carbohydrate-metabolizing enzymes in maize identified by yeast two-hybrid screeningPHYSIOLOGIA PLANTARUM, Issue 2 2005Daniela Holtgräwe We have used yeast two-hybrid screens and biochemical methods to identify glycolytic enzymes that interact with subcellular structures in hypoxic maize seedlings. As binding domain-bait fusion constructs, we have cloned actin, cytosolic aldolase, the three sucrose synthase (SUS) isoforms SUS1, SUS3, and SH1 as well as the SNF1-related protein kinase into yeast and identified cytosolic isoforms of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase, tubulin, and mitochondrial porin voltage-dependent anion channel protein (VDAC) as well as protein kinases and proteins involved in ubiquitinylation and proteasome-linked degradation as interacting activation domain-prey clones. The results were further confirmed using overlay blots (VDAC) as well as co-polymerization and co-precipitation assays (tubulin and actin). Some results were obtained that support the idea of metabolite and modification effects on the association, namely guanosine triphosphate (GTP)/MgCl2 was necessary for the binding of enolase to actin. GAPDH is inactivated upon association with tubulin but then serves to stabilize the microtubules. The findings support the idea of the dynamic formation of locally associated complexes of enzymes involved in sucrose breakdown and glycolysis in plant cells depending on their metabolic state. [source] Astroglial structures in the zebrafish brain,THE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 21 2010Larissa Grupp Abstract To understand components shaping the neuronal environment we studied the astroglial cells in the zebrafish brain using immunocytochemistry for structural and junctional markers, electron microscopy including freeze fracturing, and probed for the water channel protein aquaporin-4. Glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) showed largely overlapping immunoreactivity: GFAP in the main glial processes and GS in main processes and smaller branches. Claudin-3 immunoreactivity was spread in astroglial cells along their major processes. The ventricular lining was immunoreactive for the tight-junction associated protein ZO-1, in the telencephalon located on the dorsal, lateral, and medial surface due to the everting morphogenesis. In the tectum, subpial glial endfeet were also positive for ZO-1. Correspondingly, electron microscopy revealed junctional complexes between subpial glial endfeet. However, in freeze-fracture analysis tight junctional strands were not found between astroglial membranes, either in the optic tectum or in the telencephalon. Occurrence of aquaporin-4, the major astrocytic water channel in mammals, was demonstrated by polymerase chain reaction (PCR) analysis and immunocytochemistry in tectum and telencephalon. Localization of aquaporin-4 was not polarized but distributed along the entire radial extent of the cell. Interestingly, their membranes were devoid of the orthogonal arrays of particles formed by aquaporin-4 in mammals. Finally, we investigated astroglial cells in proliferative areas. Brain lipid basic protein, a marker of early glial differentiation but not GS, were present in some proliferation zones, whereas cells lining the ventricle were positive for both markers. Thus, astroglial cells in the zebrafish differ in many aspects from mammalian astrocytes. J. Comp. Neurol. 518:4277,4287, 2010. © 2010 Wiley-Liss, Inc. [source] Voltage-gated proton channels: what's next?THE JOURNAL OF PHYSIOLOGY, Issue 22 2008Thomas E. DeCoursey This review is an attempt to identify and place in context some of the many questions about voltage-gated proton channels that remain unsolved. As the gene was identified only 2 years ago, the situation is very different than in fields where the gene has been known for decades. For the proton channel, most of the obvious and less obvious structure,function questions are still wide open. Remarkably, the proton channel protein strongly resembles the voltage-sensing domain of many voltage-gated ion channels, and thus offers a novel approach to study gating mechanisms. Another surprise is that the proton channel appears to function as a dimer, with two separate conduction pathways. A number of significant biological questions remain in dispute, unanswered, or in some cases, not yet asked. This latter deficit is ascribable to the intrinsic difficulty in evaluating the importance of one component in a complex system, and in addition, to the lack, until recently, of a means of performing an unambiguous lesion experiment, that is, of selectively eliminating the molecule in question. We still lack a potent, selective pharmacological inhibitor, but the identification of the gene has allowed the development of powerful new tools including proton channel antibodies, siRNA and knockout mice. [source] Developmental Expression of Aquaporin 2 in the Mouse Inner Ear ,THE LARYNGOSCOPE, Issue 11 2000Michele Merves Abstract Objectives The maintenance of endolymph homeostasis is critical for the inner ear to perform its functions of hearing and maintaining balance. The identification and cloning of aquaporins (a family of water channel proteins) has allowed the study of a novel cellular mechanism potentially involved in endolymph homeostasis. The objective of the present study was to define the developmental temporal and spatial e-pression pattern of aquaporin 2 (Aqp2) in the developing mouse inner ear. Study Design A systematic immunohistochemical study of Aqp2 protein e-pression was performed on embryonic mouse inner ears ranging from embryonic day 10 (otocyst stage) to embryonic day 18 (just before birth). Methods Serial cryosections of embryonic mouse inner ears were used for immunohistochemical e-periments. A rabbit polyclonal antisera raised against a synthetic Aqp2 peptide was used with a standard nickel intensified 3,3-diaminobenzidine reaction protocol for immunolocalization of Aqp2 in tissue sections. Results Aquaporin 2 is e-pressed diffusely in the early otocyst, then becomes progressively restricted as the inner ear matures. During early cochlear duct formation (embryonic days 12 and 13), e-pression of Aqp2 is homogeneous; later, it becomes restricted to specific regions of the endolymphatic compartment (embryonic days 15 and 18). Similar restriction of e-pression patterns could be noted for the vestibular structures. Endolymphatic duct and sac and stria vascularis e-pression of Aqp2 was noted to occur fairly late during development but demonstrated a distinct pattern of immunolabeling. Conclusions Aquaporin 2 shows an early and specific pattern of e-pression in the developing mouse inner ear, suggesting a significant role for this water channel protein in the development of endolymph homeostasis and meriting further functional studies of Aqp2 in the inner ear. [source] Gene duplication, exon gain and neofunctionalization of OEP16 -related genes in land plantsTHE PLANT JOURNAL, Issue 5 2006Sinéad C. Drea Summary OEP16, a channel protein of the outer membrane of chloroplasts, has been implicated in amino acid transport and in the substrate-dependent import of protochlorophyllide oxidoreductase A. Two major clades of OEP16-related sequences were identified in land plants (OEP16-L and OEP16-S), which arose by a gene duplication event predating the divergence of seed plants and bryophytes. Remarkably, in angiosperms, OEP16-S genes evolved by gaining an additional exon that extends an interhelical loop domain in the pore-forming region of the protein. We analysed the sequence, structure and expression of the corresponding Arabidopsis genes (atOEP16-S and atOEP16-L) and demonstrated that following duplication, both genes diverged in terms of expression patterns and coding sequence. AtOEP16-S, which contains multiple G-box ABA-responsive elements (ABREs) in the promoter region, is regulated by ABI3 and ABI5 and is strongly expressed during the maturation phase in seeds and pollen grains, both desiccation-tolerant tissues. In contrast, atOEP-L, which lacks promoter ABREs, is expressed predominantly in leaves, is induced strongly by low-temperature stress and shows weak induction in response to osmotic stress, salicylic acid and exogenous ABA. Our results indicate that gene duplication, exon gain and regulatory sequence evolution each played a role in the divergence of OEP16 homologues in plants. [source] Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteinsACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2006Tarmo P. Roosild The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the `soluble' portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K+ channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86,Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments. [source] The magnocellular theory of developmental dyslexiaDYSLEXIA, Issue 1 2001John Stein Abstract Low literacy is termed ,developmental dyslexia' when reading is significantly behind that expected from the intelligence quotient (IQ) in the presence of other symptoms,incoordination, left,right confusions, poor sequencing,that characterize it as a neurological syndrome. 5,10% of children, particularly boys, are found to be dyslexic. Reading requires the acquisition of good orthographic skills for recognising the visual form of words which allows one to access their meaning directly. It also requires the development of good phonological skills for sounding out unfamiliar words using knowledge of letter sound conversion rules. In the dyslexic brain, temporoparietal language areas on the two sides are symmetrical without the normal left-sided advantage. Also brain ,warts' (ectopias) are found, particularly clustered round the left temporoparietal language areas. The visual magnocellular system is responsible for timing visual events when reading. It therefore signals any visual motion that occurs if unintended movements lead to images moving off the fovea (,retinal slip'). These signals are then used to bring the eyes back on target. Thus, sensitivity to visual motion seems to help determine how well orthographic skill can develop in both good and bad readers. In dyslexics, the development of the visual magnocellular system is impaired: development of the magnocellular layers of the dyslexic lateral geniculate nucleus (LGN) is abnormal; their motion sensitivity is reduced; many dyslexics show unsteady binocular fixation; hence poor visual localization, particularly on the left side (left neglect). Dyslexics' binocular instability and visual perceptual instability, therefore, can cause the letters they are trying to read to appear to move around and cross over each other. Hence, blanking one eye (monocular occlusion) can improve reading. Thus, good magnocellular function is essential for high motion sensitivity and stable binocular fixation, hence proper development of orthographic skills. Many dyslexics also have auditory/phonological problems. Distinguishing letter sounds depends on picking up the changes in sound frequency and amplitude that characterize them. Thus, high frequency (FM) and amplitude modulation (AM) sensitivity helps the development of good phonological skill, and low sensitivity impedes the acquisition of these skills. Thus dyslexics' sensitivity to FM and AM is significantly lower than that of good readers and this explains their problems with phonology. The cerebellum is the head ganglion of magnocellular systems; it contributes to binocular fixation and to inner speech for sounding out words, and it is clearly defective in dyslexics. Thus, there is evidence that most reading problems have a fundamental sensorimotor cause. But why do magnocellular systems fail to develop properly? There is a clear genetic basis for impaired development of magnocells throughout the brain. The best understood linkage is to the region of the Major Histocompatibility Complex (MHC) Class 1 on the short arm of chromosome 6 which helps to control the production of antibodies. The development of magnocells may be impaired by autoantibodies affecting the developing brain. Magnocells also need high amounts of polyunsaturated fatty acids to preserve the membrane flexibility that permits the rapid conformational changes of channel proteins which underlie their transient sensitivity. But the genes that underlie magnocellular weakness would not be so common unless there were compensating advantages to dyslexia. In developmental dyslexics there may be heightened development of parvocellular systems that underlie their holistic, artistic, ,seeing the whole picture' and entrepreneurial talents. Copyright © 2001 John Wiley & Sons, Ltd. [source] THIS ARTICLE HAS BEEN RETRACTED Tn5530 from Burkholderia cepacia strain 2a encodes a chloride channel protein essential for the catabolism of 2,4-dichlorophenoxyacetic acidENVIRONMENTAL MICROBIOLOGY, Issue 1 2007Antonio Sebastianelli Summary Chloride channel proteins (ClC) are found in living systems where they transport chloride ions across cell membranes. Recently, the structure/function of two prokaryotic ClC has been determined but little is known about the role of these proteins in the microbial metabolism of chlorinated compounds. Here we show that transposon Tn5530 from Burkholderia cepacia strain 2a encodes a ClC protein (BcClC) which is responsible for expelling Cl, ions generated during the catabolism of 2,4-dichlorophenoxyacetic acid (a chlorinated herbicide). We found that BcClC has the ability to transport Cl, ions across reconstituted proteoliposome membranes. We created two mutants in which the intrachannel glutamate residue of the protein, known to be responsible for opening and closing the channel (i.e. gating), was changed in order to create constitutively open and closed forms. We observed that cells carrying the closed-channel protein accumulated Cl, ions intracellularly leading to a decrease in intracellular pH, cell stasis and death. Further, we established that BcClC has the same gating mechanism as that reported for the ClC protein from Salmonella typhimurium. Our results show that the physiological role of ClC is to maintain cellular homeostasis which can be impaired by the catabolism of chlorinated compounds. [source] Biotic ligand model of the acute toxicity of metals.ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 10 2001Abstract The biotic ligand model (BLM) of acute metal toxicity to aquatic organisms is based on the idea that mortality occurs when the metal,biotic ligand complex reaches a critical concentration. For fish, the biotic ligand is either known or suspected to be the sodium or calcium channel proteins in the gill surface that regulate the ionic composition of the blood. For other organisms, it is hypothesized that a biotic ligand exists and that mortality can be modeled in a similar way. The biotic ligand interacts with the metal cations in solution. The amount of metal that binds is determined by a competition for metal ions between the biotic ligand and the other aqueous ligands, particularly dissolved organic matter (DOM), and the competition for the biotic ligand between the toxic metal ion and the other metal cations in solution, for example, calcium. The model is a generalization of the free ion activity model that relates toxicity to the concentration of the divalent metal cation. The difference is the presence of competitive binding at the biotic ligand, which models the protective effects of other metal cations, and the direct influence of pH. The model is implemented using the Windermere humic aqueous model (WHAM) model of metal,DOM complexation. It is applied to copper and silver using gill complexation constants reported by R. Playle and coworkers. Initial application is made to the fathead minnow data set reported by R. Erickson and a water effects ratio data set by J. Diamond. The use of the BLM for determining total maximum daily loadings (TMDLs) and for regional risk assessments is discussed within a probabilistic framework. At first glance, it appears that a large amount of data are required for a successful application. However, the use of lognormal probability distributions reduces the required data to a manageable amount. [source] Aquaporin 4 changes in rat brain with severe hydrocephalusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006Xiaoyan Mao Abstract Hydrocephalus is characterized by impaired cerebrospinal fluid (CSF) flow with enlargement of the ventricular cavities of the brain and progressive damage to surrounding tissue. Bulk water movement is altered in these brains. We hypothesized that increased expression of aquaporins, which are water-permeable channel proteins, would occur in these brains to facilitate water shifts. We used quantitative (real-time) RT-PCR, Western blotting and immunohistochemistry to evaluate the brain expression of aquaporins (AQP) 1, 4, and 9 mRNA and protein in Sprague,Dawley rats rendered hydrocephalic by injection of kaolin into cistern magna. AQP4 mRNA was significantly up-regulated in parietal cerebrum and hippocampus 4 weeks and 9 months after induction of hydrocephalus (P < 0.05). Although Western blot analysis showed no significant change, there was more intense perivascular AQP4 immunoreactivity in cerebrum of hydrocephalic brains at 3,4 weeks after induction. We did not detect mRNA or protein changes in AQP1 (located in choroid plexus) or AQP9 (located in select neuron populations). Kir4.1, a potassium channel protein linked to water flux, exhibited enhanced immunoreactivity in the cerebral cortex of hydrocephalic rats; the perineuronal distribution was entirely different from that of AQP4. These results suggest that brain AQP4 up-regulation might be a compensatory response to maintain water homeostasis in hydrocephalus. [source] Exercise training attenuates ageing-induced BKCa channel downregulation in rat coronary arteriesEXPERIMENTAL PHYSIOLOGY, Issue 6 2010Sulayma Albarwani Physical inactivity and ageing are widely recognized as risk factors for development of coronary artery disease. One of the characteristic changes that occurs in aged coronary artery is downregulation of their large-conductance voltage- and calcium-activated K+ (BKCa) channels. In this study, we investigated the effects of moderate exercise training (ET) on the activity of BKCa channels in coronary arteries of aged rats. Old Fischer 344 rats (23,26 months old) were randomly assigned to sedentary (O-SED, n= 24) or exercise-trained groups (O-ET, n= 28). The O-ET rats underwent a progressive treadmill exercise-training programme for 60 min day,1, 5 days week,1 for 12 weeks. Young animals were used for comparison. Coronary arteries were mounted on a wire myograph, and contractions in response to 1, 10, 30, 50 and 100 nmol l,1 iberiotoxin were compared. Iberiotoxin (100 nmol l,1) contracted coronary arteries of young, O-SED and O-ET rats by 115 ± 14, 36 ± 5.6 and 61 ± 5% of 5-hydroxytryptamine-induced contractions, respectively. Patch-clamp studies revealed a larger magnitude of BKCa current in young (104 ± 15.6 pA pF,1) compared with O-ET (44 ± 9 pA pF,1) and least in O-SED coronary smooth muscle cells (8.6 ± 2 pA pF,1). Western immunoblotting was performed to study expression levels of BKCa channel proteins. The , and ,1 subunits of the BKCa channel were reduced by 40 ± 3.5 and 30 ± 2.6%, respectively, in coronary arteries of old compared with young rats, and ET attenuated this reduction in expression level to 28 ± 2 and 12 ± 4%, respectively. Our results showed that ageing was associated with a reduction in BKCa channels, and ET partly reversed this reduction. We conclude that low-intensity ET may be beneficial in restoring age-related decline in coronary vasodilatory properties mediated by BKCa channels. [source] Crystal structure of the soluble form of the redox-regulated chloride ion channel protein CLIC4FEBS JOURNAL, Issue 19 2005Dene R. Littler The structure of CLIC4, a member of the CLIC family of putative intracellular chloride ion channel proteins, has been determined at 1.8 Å resolution by X-ray crystallography. The protein is monomeric and it is structurally similar to CLIC1, belonging to the GST fold class. Differences between the structures of CLIC1 and CLIC4 are localized to helix 2 in the glutaredoxin-like N-terminal domain, which has previously been shown to undergo a dramatic structural change in CLIC1 upon oxidation. The structural differences in this region correlate with the sequence differences, where the CLIC1 sequence appears to be atypical of the family. Purified, recombinant, wild-type CLIC4 is shown to bind to artificial lipid bilayers, induce a chloride efflux current when associated with artificial liposomes and produce an ion channel in artificial bilayers with a conductance of 30 pS. Membrane binding is enhanced by oxidation of CLIC4 while no channels were observed via tip-dip electrophysiology in the presence of a reducing agent. Thus, recombinant CLIC4 appears to be able to form a redox-regulated ion channel in the absence of any partner proteins. [source] Genotype-dependent sensitivity of hepatitis C virus to inhibitors of the p7 ion channel,HEPATOLOGY, Issue 6 2008Stephen Griffin The hepatitis C virus (HCV) p7 protein plays a critical role during particle formation in cell culture and is required for virus replication in chimpanzees. The discovery that it displayed cation channel activity in vitro led to its classification within the "viroporin" family of virus-coded ion channel proteins, which includes the influenza A virus (IAV) M2 protein. Like M2, p7 was proposed as a potential target for much needed new HCV therapies, and this was supported by our finding that the M2 inhibitor, amantadine, blocked its activity in vitro. Since then, further compounds have been shown to inhibit p7 function but the relationship between inhibitory effects in vitro and efficacy against infectious virus is controversial. Here, we have sought to validate multiple p7 inhibitor compounds using a parallel approach combining the HCV infectious culture system and a rapid throughput in vitro assay for p7 function. We identify a genotype-dependent and subtype-dependent sensitivity of HCV to p7 inhibitors, in which results in cell culture largely mirror the sensitivity of recombinant protein in vitro; thus building separate sensitivity profiles for different p7 sequences. Inhibition of virus entry also occurred, suggesting that p7 may be a virion component. Second site effects on both cellular and viral processes were identified for several compounds in addition to their efficacy against p7 in vitro. Nevertheless, for some compounds antiviral effects were specific to a block of ion channel function. Conclusion: These data validate p7 inhibitors as prototype therapies for chronic HCV disease. (HEPATOLOGY 2008;48:1779-1790.) [source] Chronic constriction injury induces aquaporin-2 expression in the dorsal root ganglia of ratsJOURNAL OF ANATOMY, Issue 5 2009Barbara Buffoli Abstract Aquaporins are a family of water channel proteins involved in water homeostasis in several tissues. Current knowledge of aquaporin expression in the nervous system is very limited. Therefore the first aim of this study was to assess, by immunohistochemistry and immunoblotting analysis, the presence and localization of aquaporin-2 in the spinal cord and dorsal root ganglia of naïve adult rats. In addition, we evaluated aquaporin-2 expression in response to chronic constriction injury of the sciatic nerve, a model of neuropathic pain. Our results showed that aquaporin-2 expression was not detectable either in the spinal cord or the dorsal root ganglia of naïve rats. However, we showed for the first time an increase of aquaporin-2 expression in response to chronic constriction injury treatment in small-diameter dorsal root ganglia neurons but no expression in the lumbar spinal cord. These data support the hypothesis that aquaporin-2 expression is involved in inflammatory neuropathic nerve injuries, although its precise role remains to be determined. [source] Molecular and cellular mechanisms of neuroprotection by vascular endothelial growth factorJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1-2 2005Feng-Yan Sun Abstract The present view of the neuroprotective functions and mechanisms of action of vascular endothelial growth factor (VEGF) is based on studies of neuronal ischemic/hypoxic models in vivo and in vitro. Endogenous neuronal VEGF increases in the ischemic brain and plays a neuroprotective role in the pathophysiologic processes that follow stroke. Exogenous VEGF, directly administered or overexpressed by gene delivery into rat brains, reduces ischemic brain infarct and decreases hypoxic neuronal death. The main neuroprotective mechanisms of VEGF include: (1) modulation of the phosphatidylinositol 3,-kinase (PI3K)/Akt/nuclear factor-,B signaling pathway, inhibition of caspase-3 activity, and reduction of ischemic neuronal apoptosis; (2) inhibition of outward delayed rectifier potassium channel currents and increase of ischemia-induced tyrosine phosphorylation of Kv1.2 potassium channel proteins via activation of the PI3K pathway; and (3) enhancement of proliferation and migration of neural progenitors in the subventricular zone and improvement of striatal neurogenesis and maturation of newborn neurons in adult rat brains after stroke. © 2004 Wiley-Liss, Inc. [source] Ternary complex formation between AmtB, GlnZ and the nitrogenase regulatory enzyme DraG reveals a novel facet of nitrogen regulation in bacteriaMOLECULAR MICROBIOLOGY, Issue 6 2007Luciano F. Huergo Summary Ammonium movement across biological membranes is facilitated by a class of ubiquitous channel proteins from the Amt/Rh family. Amt proteins have also been implicated in cellular responses to ammonium availability in many organisms. Ammonium sensing by Amt in bacteria is mediated by complex formation with cytosolic proteins of the PII family. In this study we have characterized in vitro complex formation between the AmtB and PII proteins (GlnB and GlnZ) from the diazotrophic plant-associative bacterium Azospirillum brasilense. AmtB,PII complex formation only occurred in the presence of adenine nucleotides and was sensitive to 2-oxoglutarate when Mg2+ and ATP were present, but not when ATP was substituted by ADP. We have also shown in vitro complex formation between GlnZ and the nitrogenase regulatory enzyme DraG, which was stimulated by ADP. The stoichiometry of this complex was 1:1 (DraG monomer : GlnZ trimer). We have previously reported that in vivo high levels of extracellular ammonium cause DraG to be sequestered to the cell membrane in an AmtB and GlnZ-dependent manner. We now report the reconstitution of a ternary complex involving AmtB, GlnZ and DraG in vitro. Sequestration of a regulatory protein by the membrane-bound AmtB,PII complex defines a new regulatory role for Amt proteins in Prokaryotes. [source] How do membrane proteins sense water stress?MOLECULAR MICROBIOLOGY, Issue 4 2002Bert Poolman Summary Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmo-protectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift-activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein,lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift-activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins. [source] | ||||||||||||||||||||||||||||||||||