Analyse fonctionnelle de deux nouvelles mutations récessives de l’AQP2 impliquées dans le diabète insipide néphrogénique par expression dans les ovocytes de Xenopus laevis

Le diabète insipide néphrogénique (DIN) autosomal peut être causé par les mutations du gène codant pour le canal à eau aquaporine-2 (AQP2). Un modèle couramment utilisé pour l’étude des protéines membranaires telle l’AQP2 est l’expression hétérologue dans les ovocytes de Xenopus laevis. Malheureusement, les techniques déjà existantes de purification de membranes plasmiques sont soit trop longues, trop difficiles ou demandent trop de matériel, ne permettent pas l’analyse adéquate du ciblage des formes sauvage comme mutantes, un élément crucial de ce type d’étude. Nous avons donc dans un premier temps mis au point une technique rapide et efficace de purification de membranes plasmiques qui combine la digestion partielle de la membrane vitelline, sa polymérisation à la membrane plasmique suivi de centrifugations à basse vitesse pour récolter les membranes purifiées. Nous avons utilisé cette technique dans l’étude de deux nouveaux cas familiaux de patients hétérozygotes possédant les mutations V24A et R187C dans un cas et K228E et R187C dans le second cas. Pour chaque mutation, nous avons analysé autant les éléments de fonctionnalité que les paramètres d’expression des protéines mutantes. Les expériences de perméabilité membranaire démontrent que les ovocytes exprimant AQP2-V24A (Pf = 16.3 ± 3.5 x 10-4 cm/s, 10 ng) et AQP2- K228E (Pf = 19.9 ± 7.0 x 10-4 cm/s, 10 ng) ont des activités similaires à celle exprimant la forme native (Pf = 14.4 ± 5.5 x 10-4 cm/s, 1 ng), tandis que AQP2- R187C (Pf = 2.6 ± 0.6 x 10-4 cm/s, 10 ng) ne semble avoir aucune activité comme ce qui est observé chez les ovocytes non-injectés (Pf = 2.8 ± 1.0 x 10-4 cm/s). Les études de co-expression ont démontré un effet d’additivité lorsque AQP2-V24A et -K228E sont injectées avec la forme native et un effet s’apparentant à la dominance négative lorsque AQP2-R187C est injecté avec la forme native, avec AQP2-V24A ou avec –K228E. Les résultats obtenus par immunobuvardage représente bien ce qui a été démontré précédemment, on remarque la présence des mutations K228E, V24A et la forme sauvage à la membrane plasmique, contrairement à la mutation R187C. Cependant, lorsque les mutations sont exprimées dans des cellules mIMCD-3, il n’y a qu’une faible expression à la membrane de la forme –K228E et une absence totale des formes –V24A et –R187C à la membrane plasmique, contrairement à la forme native. Les résultats de nos études démontrent que tout dépendant du système d’expression les formes –K228E et –V24A peuvent être utiles dans l’étude des problèmes d’adressage à la membrane à l’aide de chaperonne chimique. De plus, la forme –R187C démontre des difficultés d’adressage qui devront être étudiées afin de mieux comprendre la synthèse des formes natives.

Aquaporin 9 (AQP9) localization in the adult dog testis excurrent ducts by immunohistochemistry

Aquaporins (AQPs) are small, intrinsic membrane proteins that are present in many cell types involved in fluid transport. AQP9 is a major apical water channel that is expressed throughout the efferent ducts, epididymis, and vas deferens, as well as in other regions of the human and rodent male reproductive tract. The target of this study was to examine the expression of AQP9 in epithelial cells in the adult dog efferent ducts, epididymis, and vas deferens. Samples of dog male reproductive tract comprising fragments of the testis; initial segment, caput, corpus, and cauda of the epididymis; and vas deferens were obtained from eight adult mongrel dogs. Immunohistochemistry and Western blotting procedures were used to show AQP9 localization and distribution. AQP9 expression was not detected either in dog seminiferous tubules or rete testis. However, apical labeling for AQP9 was detected in the different regions of epididymis and vas deferens, with the reaction being less intense in the caput epididymis. Thus, AQP9 is abundantly expressed in dog male reproductive tract, in which it is an important apical pathway for transmembrane flow of water and neutral solutes.

Immunolocalization of aquaporins 1, 2 and 7 in rete testis, efferent ducts, epididymis and vas deferens of adult dog

The transepithelial movement of water into the male reproductive tract is an essential process for normal male fertility. Protein water channels, referred to as aquaporins (AQPs), are involved in increasing the osmotic permeability of membranes. This study has examined the expression of AQP1, AQP2, and AQP7 in epithelial cells in adult dog efferent ducts, epididymis, and vas deferens. Samples of dog male reproductive tract comprising fragments of the testis, initial segment, caput, corpus and cauda epididymidis, and vas deferens were investigated by immunohistochemistry and Western blotting procedures to show the localization and distribution of the AQPs. AQP1 was noted in rete testis, in efferent ducts, and in vessels in the intertubular space, suggesting that AQP1 participated in the absorption of the large amount of testicular fluid occurring characteristically in the efferent ducts. AQP2 expression was found in the rete testis, efferent ducts and epididymis, whereas AQP7 was expressed in the epithelium of the proximal regions of the epididymis and in the vas deferens. This is the first time that AQP2 and AQP7 have been observed in these regions of mammalian excurrent ducts, but their functional role in the dog male reproductive tract remains unknown. Investigations of AQP biology could be relevant for clinical studies of the male reproductive tract and to technologies for assisted procreation.

Myasthenia gravis and neuromyelitis optica spectrum disorder A multicenter study of 16 patients

Objective: To describe 16 patients with a coincidence of 2 rare diseases: aquaporin-4 antibody (AQP4-Ab)-mediated neuromyelitis optica spectrum disorder (AQP4-NMOSD) and acetylcholine receptor antibody (AChR-Ab)-mediated myasthenia gravis (AChR-MG). Methods: The clinical details and antibody results of 16 patients with AChR-MG and AQP4-NMOSD were analyzed retrospectively. Results: All had early-onset AChR-MG, the majority with mild generalized disease, and a high proportion achieved remission. Fifteen were female; 11 were Caucasian. In 14/16, the MG preceded NMOSD (median interval: 16 years) and 11 of these had had a thymectomy although 1 only after NMOSD onset. In 4/5 patients tested, AQP4-Abs were detectable between 4 and 16 years prior to disease onset, including 2 patients with detectable AQP4-Abs prior to thymectomy. AChR-Abs decreased and the AQP4-Ab levels increased over time in concordance with the relevant disease. AChR-Abs were detectable at NMOSD onset in the one sample available from 1 of the 2 patients with NMOSD before MG. Conclusions: Although both conditions are rare, the association of MG and NMOSD occurs much more frequently than by chance and the MG appears to follow a benign course. AChR-Abs or AQP4-Abs may be present years before onset of the relevant disease and the antibody titers against AQP4 and AChR tend to change in opposite directions. Although most cases had MG prior to NMOSD onset, and had undergone thymectomy, NMOSD can occur first and in patients who have not had their thymus removed. Neurology (R) 2012;78:1601-1607

Protein-Protein- und Protein-Lipid-Wechselwirkungen beeinflussen die Oligomerisierung und Funktion des E. coli Aquaglyceroporins GlpF

Aquaporine sind hochselektive Transmembrankanäle, die in allen Lebensformen den Fluss von Wasser und kleinen, polaren Molekülen wie Glycerol über Lipidmembranen ermöglichen. Obwohl die Kanalpore für den Substratfluss im Monomer lokalisiert ist, liegen Aquaporine innerhalb biologischer Membranen als Homotetramere vor. Im Rahmen dieser Arbeit wurden proteinbezogene und lipidmembranassoziierte Einflüsse auf die Oligomerisierung und Funktion des bakteriellen Aquaglyceroporins GlpF sowohl in vitro als auch in vivo untersucht. rnDie erhöhte Stabilität der Aquaporinpore sowie Interaktion zwischen den GlpF-Monomeren sind Triebkräfte der Aquaporin-Tetramerisierung. Ferner erfordern die GlpF-Tetramerisierung und -Aktivität bei Abschirmung der Ladung anionischer Lipide und einer minimalen Membrandicke von 27 Å keine spezielle Lipidumgebung. Da anionische Lipide die GlpF-Funktion jedoch störten, kann die GlpF-Aktivität in vivo möglicherweise durch die selektive Anreicherung von anionischen Lipiden in der unmittelbaren Proteinumgebung reguliert werden. Ungünstige Lipid-GlpF-Interaktionen können jedoch in Lipidumgebungen mit hoher Ordnung in der Acylkettenregion entstehen, die zu einer Aggregation der GlpF-Tetramere und reduzierten Aktivität führen. rnFerner wurde die Auswirkung der nephrogenen Diabetes insipidus verursachenden Aquaporin 2-Punktmutation V71M auf die Oligomerisierung und Funktion des homologen, bakteriellen Aquaglyceroporins GlpF untersucht. Da weder die Oligomierisierung noch die Aktivität des homologen, bakteriellen Aquaglyceroporins eingeschränkt sind, beruht der Krankheitsmechanismus der Aquaporin 2-Mutante V71M vermutlich auf einem defekten Transportmechansimus im Menschen. rn

Loss of astrocyte polarization upon transient focal brain ischemia as a possible mechanism to counteract early edema formation

Brain edema is the main cause of death from brain infarction. The polarized expression of the water channel protein aquaporin-4 (AQP4) on astroglial endfeet surrounding brain microvessels suggests a role in brain water balance. Loss of astrocyte foot process anchoring to the basement membrane (BM) accompanied by the loss of polarized localization of AQP4 to astrocytic endfeet has been shown to be associated with vasogenic/extracellular edema in neuroinflammation. Here, we asked if loss of astrocyte polarity is also observed in cytotoxic/intracellular edema following focal brain ischemia after transient middle cerebral artery occlusion (tMCAO). Upon mild focal brain ischemia, we observed diminished immunostaining for the BM components laminin α4, laminin α2, and the proteoglycan agrin, in the core of the lesion, but not in BMs in the surrounding penumbra. Staining for the astrocyte endfoot anchorage protein β-dystroglycan (DG) was dramatically reduced in both the lesion core and the penumbra, and AQP4 and Kir4.1 showed a loss of polarized localization to astrocytic endfeet. Interestingly, we observed that mice deficient for agrin expression in the brain lack polarized localization of β-DG and AQP4 at astrocytic endfeet and do not develop early cytotoxic/intracellular edema following tMCAO. Taken together, these data indicate that the binding of DG to agrin embedded in the subjacent BM promotes polarized localization of AQP4 to astrocyte endfeet. Reduced DG protein levels and redistribution of AQP4 as observed upon tMCAO might therefore counteract early edema formation and reflect a beneficial mechanism operating in the brain to minimize damage upon ischemia.

Loss of astrocyte polarity marks blood-brain barrier impairment during experimental autoimmune encephalomyelitis

In multiple sclerosis (MS), and its animal model experimental autoimmune encephalomyelitis (EAE), dysfunction of the blood-brain barrier (BBB) leads to edema formation within the central nervous system. The molecular mechanisms of edema formation in EAE/MS are poorly understood. We hypothesized that edema formation is due to imbalanced water transport across the BBB caused by a disturbed crosstalk between BBB endothelium and astrocytes. Here, we demonstrate at the light microscopic and ultrastructural level, the loss of polarized localization of the water channel protein aquaporin-4 (AQP4) in astrocytic endfeet surrounding microvessels during EAE. AQP4 was found to be redistributed over the entire astrocytic cell surface and lost its arrangement in orthogonal arrays of intramembranous particles as seen in the freeze-fracture replica. In addition, immunostaining for the astrocytic extracellular matrix receptor beta-dystroglycan disappeared from astroglial membranes in the vicinity of inflammatory cuffs, whereas immunostaining for the dystroglycan ligands agrin and laminin in the perivascular basement membrane remained unchanged. Our data suggest that during EAE, loss of beta-dystroglycan-mediated astrocyte foot process anchoring to the basement membrane leads to loss of polarized AQP4 localization in astrocytic endfeet, and thus to edema formation in EAE.

Agrin defines polarized distribution of orthogonal arrays of particles in astrocytes

Accumulating evidence indicates that agrin, a heparan sulphate proteoglycan of the extracellular matrix, plays a role in the organization and maintenance of the blood-brain barrier. This evidence is based on the differential effects of agrin isoforms on the expression and distribution of the water channel protein, aquaporin-4 (AQP4), on the swelling capacity of cultured astrocytes of neonatal mice and on freeze-fracture data revealing an agrin-dependent clustering of orthogonal arrays of particles (OAPs), the structural equivalent of AQP4. Here, we show that the OAP density in agrin-null mice is dramatically decreased in comparison with wild-types, by using quantitative freeze-fracture analysis of astrocytic membranes. In contrast, anti-AQP4 immunohistochemistry has revealed that the immunoreactivity of the superficial astrocytic endfeet of the agrin-null mouse is comparable with that in wild-type mice. Moreover, in vitro, wild-type and agrin-null astrocytes cultured from mouse embryos at embryonic day 19.5 differ neither in AQP4 immunoreactivity, nor in OAP density in freeze-fracture replicas. Analyses of brain tissue samples and cultured astrocytes by reverse transcription with the polymerase chain reaction have not demonstrated any difference in the level of AQP4 mRNA between wild-type astrocytes and astrocytes from agrin-null mice. Furthermore, we have been unable to detect any difference in the swelling capacity between wild-type and agrin-null astrocytes. These results clearly demonstrate, for the first time, that agrin plays a pivotal role for the clustering of OAPs in the endfoot membranes of astrocytes, whereas the mere presence of AQP4 is not sufficient for OAP clustering.

Experiment: Dissecting the impact of CO2 and pH on the mechanisms of photosynthesis and calcification in the coccolithophore Emiliania huxleyi

Coccolithophores are important calcifying phytoplankton predicted to be impacted by changes in ocean carbonate chemistry caused by the absorption of anthropogenic CO2. However, it is difficult to disentangle the effects of the simultaneously changing carbonate system parameters (CO2, bicarbonate, carbonate and protons) on the physiological responses to elevated CO2. Here, we adopted a multifactorial approach at constant pH or CO2 whilst varying dissolved inorganic carbon (DIC) to determine physiological and transcriptional responses to individual carbonate system parameters. We show that Emiliania huxleyi is sensitive to low CO2 (growth and photosynthesis) and low bicarbonate (calcification) as well as low pH beyond a limited tolerance range, but is much less sensitive to elevated CO2 and bicarbonate. Multiple up-regulated genes at low DIC bear the hallmarks of a carbon-concentrating mechanism (CCM) that is responsive to CO2 and bicarbonate but not to pH. Emiliania huxleyi appears to have evolved mechanisms to respond to limiting rather than elevated CO2. Calcification does not function as a CCM, but is inhibited at low DIC to allow the redistribution of DIC from calcification to photosynthesis. The presented data provides a significant step in understanding how E. huxleyi will respond to changing carbonate chemistry at a cellular level

Altered ubiquitination and stability of aquaporin-1 in hypertonic stress

Aquaporin-1 (AQP1) water channel protein expression is increased by hypertonic stress. The contribution of changes in protein stability to hypertonic induction of AQP1 have not been described. Incubation of BALB/c fibroblasts spontaneously expressing AQP1 with proteasome inhibitors increased AQP1 expression, suggesting basal proteasome-dependent degradation of the protein. Degradation by the proteasome is thought to be triggered by polyubiquitination of a target protein. To determine whether AQP1 is ubiquitinated, immunoprecipitation with anti-AQP1 antibodies was performed, and the resultant samples were probed by protein immunoblot for the presence of ubiquitin. Immunoblots demonstrated ubiquitination of AQP1 under control conditions that increased after treatment with proteasome inhibitors (MG132, lactacystin). Exposure of cells to hypertonic medium for as little as 4 h decreased ubiquitination of AQP1, an effect that persisted through 24 h in hypertonic medium. Using metabolic labeling with [35S]methionine, the half-life of AQP1 protein under isotonic conditions was found to be <4 h. AQP1 protein half-life was markedly increased by exposure of cells to hypertonic medium. These observations provide evidence that aquaporins are a target for ubiquitination and proteasome-dependent degradation. Additionally, these studies demonstrate that reduced protein ubiquitination and increased protein stability lead to increased levels of AQP1 expression during hypertonic stress.

High Expression of the Tonoplast Aquaporin ZmTIP1 in Epidermal and Conducting Tissues of Maize1

Aquaporins are integral membrane proteins of the tonoplast and the plasma membrane that facilitate the passage of water through these membranes. Because of their potentially important role in regulating water flow in plants, studies documenting aquaporin gene expression in specialized tissues involved in water and solute transport are important. We used in situ hybridization to examine the expression pattern of the tonoplast aquaporin ZmTIP1 in different organs of maize (Zea mays L.). This tonoplast water channel is highly expressed in the root epidermis, the root endodermis, the small parenchyma cells surrounding mature xylem vessels in the root and the stem, phloem companion cells and a ring of cells around the phloem strand in the stem and the leaf sheath, and the basal endosperm transfer cells in developing kernels. We postulate that the high level of expression of ZmTIP1 in these tissues facilitates rapid flow of water through the tonoplast to permit osmotic equilibration between the cytosol and the vacuolar content, and to permit rapid transcellular water flow through living cells when required.

Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney.

The renal urea transporter (RUT) is responsible for urea accumulation in the renal medulla, and consequently plays a central role in the urinary concentrating mechanism. To study its cellular and subcellular localization, we prepared affinity-purified, peptide-derived polyclonal antibodies against rat RUT based on the cloned cDNA sequence. Immunoblots using membrane fractions from rat renal inner medulla revealed a solitary 97-kDa band. Immunocytochemistry demonstrated RUT labeling of the apical and subapical regions of inner medullary collecting duct (IMCD) cells, with no labeling of outer medullary or cortical collecting ducts. Immunoelectron microscopy directly demonstrated labeling of the apical plasma membrane and of subapical intracellular vesicles of IMCD cells, but no labeling of the basolateral plasma membrane. Immunoblots demonstrated RUT labeling in both plasma membrane and intracellular vesicle-enriched membrane fractions from inner medulla, a subcellular distribution similar to that of the vasopressin-regulated water channel, aquaporin-2. In the outer medulla, RUT labeling was seen in terminal portions of short-loop descending thin limbs. Aside from IMCD and descending thin limbs, no other structures were labeled in the kidney. These results suggest that: (i) the RUT provides the apical pathway for rapid, vasopressin-regulated urea transport in the IMCD, (ii) collecting duct urea transport may be increased by vasopressin by stimulation of trafficking of RUT-containing vesicles to the apical plasma membrane, and (iii) the rat urea transporter may provide a pathway for urea entry into the descending limbs of short-loop nephrons.

Unusual 1,6-diphenyl-1,3,5-hexatriene (DPH) spectrophotometric behavior in water/ethanol and water/DMSO mixtures

The absorption spectra of DPH at fixed concentration do not change with water content in organic solvents. It exhibits monomer bands, such as those obtained in ethanol. The absorption did not change for solutions up to 54 and 46% of water in ethanol and DMSO, respectively, for [DPH] = 5.0 × 10-6 mol L-1 at 30 °C. However, at the same experimental conditions, a gradual sharp decay of the DPH fluorescence is observed. It is proposed that water molecules below these water concentration limits act as quenchers of the excited states of DPH. Stern-Volmer quenching constants by intensities measurements are 7.4 × 10-2 (water/ethanol) and 2.6 × 10-2 L mol-1 (water/DMSO). DPH lifetime measurements in the absence and presence of water resulted in 7.1 × 10-2 L mol-1 in water/ethanol, which pointed out that the process is a dynamic quenching by water molecules. For experiments using DPH as probe, this process can affect data, leading to misunderstanding interpretation.

Experimental investigation of flow-induced vibration on isolated and tandem circular cylinders fitted with strakes

The effect of varying the geometric parameters of helical strakes on vortex-induced vibration (VIV) is investigated in this paper. The degree of oscillation attenuation or even suppression is analysed for isolated circular cylinder cases. How a cylinder fitted with strakes behaves when immersed in the wake of another cylinder in tandem arrangement is also investigated and these results are compared to those with a single straked cylinder. The experimental tests are conducted at a circulating water channel facility and the cylindrical models are mounted on a low-damping air bearing elastic base with one degree-of-freedom, restricted to oscillate in the transverse direction to the channel flow. Three strake pitches (p) and heights (h) are tested: p = 5, 10, 15d, and h = 0.1, 0.2, 0.25d. The mass ratio is 1.8 for all models. The Reynolds number range is from 1000 to 10000, and the reduced velocity varies up to 21. The cases with h = 0.1d strakes reduce the amplitude response when compared to the isolated plain cylinder, however the oscillation still persists. On the other hand, the cases with h = 0.2, 0.25d strakes almost completely suppress VIV. Spanwise vorticity fields, obtained through stereoscopic digital particle image velocimetry (SDPIV), show an alternating vortex wake for the p = 10d and h = 0.1d straked cylinder. The p = 10d and h = 0.2d cylinder wake has separated shear layers with constant width and no roll-up close to the body. The strakes do not increase the magnitude of the out-of-plane velocity compared to the isolated plain cylinder. However, they deflect the flow in the out-of-plane direction in a controlled way, which can prevent the vortex shedding correlation along the span. In order to investigate the wake interference effect on the strake efficiency, an experimental arrangement with two cylinders in tandem is employed. The centre-to-centre distance for the tandem arrangement varies from 2 to 6. When the downstream p = 10d and h = 0.2d cylinder is immersed in the wake of an upstream fixed plain cylinder, it loses its effectiveness compared with the isolated case. Although the oscillations have significant amplitude, they are limited, which is a different behaviour from that of a tandem configuration with two plain cylinders. For this particular case, the amplitude response monotonically increases for all gaps, except one, a trait usually found in galloping-like oscillations. SDPIV results for the tandem arrangements show alternating vortex shedding and oscillatory wake. (C) 2010 Elsevier Ltd. All rights reserved.

HLA-DRB1*1501 tagging rs3135388 polymorphism is not associated with neuromyelitis optica

Background: Association of the HLA-DRB1*1501 allele with multiple sclerosis is well established, but its association with neuromyelitis optica has only been evaluated in small populations. Methods: We performed a case-control genetic association study to evaluate the association of HLA-DRB1*1501 with neuromyelitis optica. The single nucleotide polymorphism rs3135388, which tags HLA-DRB1*1501, was genotyped in 164 patients with neuromyelitis optica, 220 patients with multiple sclerosis and 959 controls matched for age, gender and ethnicity. Genotyping for rs3135388 was performed by Taqman-based 5` nuclease assay. Results: Rs3135388*A was positively associated with multiple sclerosis (OR = 3.93; 95% CI = 2.58-5.97, p = 1.18 x 10(-09)) but negatively associated with NMO (OR = 0.57; 95% CI = 0.36-0.91, p = 0.01). Conclusions: Multiple sclerosis and neuromyelitis optica differ in their associations with DRB1*1501.