Deletion of beaded filament proteins or the C-terminal end of Aquaporin 0 causes analogous abnormal distortion aberrations in mouse lens.

Lens-specific beaded filament (BF) proteins CP49 and filensin interact with the C-terminus of the water channel protein Aquaporin 0 (AQP0). Previously we have reported that a C-terminally end-deleted AQP0-expressing transgenic mouse model AQP0ΔC/ΔC developed abnormal optical aberrations in the lens. This investigation was undertaken to find out whether the total loss of the BF structural proteins alter the optical properties of the lens and cause optical aberrations similar to those in AQP0ΔC/ΔC lenses; also, to map the changes in the optical quality as a function of age in the single or double BF protein knockouts as well as to assess whether there is any significant change in the water channel function of AQP0 in these knockouts. A double knockout mouse (2xKO) model for CP49 and filensin was developed by crossing CP49-KO and filensin-KO mice. Wild type, CP49-KO, filensin-KO, and 2xKO lenses at different ages, and AQP0ΔC/ΔC lenses at postnatal day-17 were imaged through the optical axis and compared for optical quality and focusing property. All three knockout models showed loss of transparency, and development of abnormal optical distortion aberration similar to that in AQP0ΔC/ΔC. Copper grid focusing by the lenses at 6, 9 and 12 months of age showed an increase in aberrations as age advanced. With progression in age, the grid images produced by the lenses of all KO models showed a transition from a positive barrel distortion aberration to a pincushion distortion aberration with the formation of three distinct aberration zones similar to those produced by AQP0ΔC/ΔC lenses. Water permeability of fiber cell membrane vesicles prepared from CP49-KO, filensin-KO and 2xKO models, measured using the osmotic shrinking method, remained similar to that of the wild type without any statistically significant alteration (P > 0.05). Western blotting and quantification revealed the expression of comparable quantities of AQP0 in all three BF protein KOs. Our study reveals that loss of single or both beaded filament proteins significantly affect lens refractive index gradient, transparency and focusing ability in an age-dependent manner and the interaction of BF proteins with AQP0 is critical for the proper functioning of the lens. The presence of BF proteins is necessary to prevent abnormal optical aberrations and maintain homeostasis in the aging lens.

Enhanced ammonia detoxification to urea in hepatocytes transduced with human aquaporin-8 gene.

Hepatic ammonia detoxification to urea is critical for the prevention of hyperammonemia and neurological damage. Hepatocyte mitochondrial aquaporin-8 (AQP8) channels have been involved in ammonia-derived ureagenesis. Herein, we studied whether the adenoviral gene transfer of human AQP8 (hAQP8) to hepatocyte mitochondria enhances ammonia conversion to urea. Using primary cultured rat hepatocytes, we first confirmed the mitochondrial expression of hAQP8 and then, using unlabeled or 15 N-labeled ammonia, we demonstrated that the urea synthesis was significantly enhanced in hAQP8-transduced hepatocytes. Studies using isolated hAQP8-expressing mitochondria also showed an increased ammonia metabolism. hAQP8 transduction was able to recover the impaired ammonia-derived ureagenesis in hepatotoxin-treated hepatocytes. Our data suggest that mitochondrially-expressed hAQP8 enhances and improves hepatocyte ammonia conversion to urea, a finding with potential therapeutic implications for liver disease with impaired ammonia detoxification.

An ABA Functional Analogue B2 Enhanced Salt Tolerance by Inducing the Root Elongation and Reducing Peroxidation Damage in Maize Seedlings.

Salt stress negatively affects maize growth and yield. Application of plant growth regulator is an effective way to improve crop salt tolerance, therefore reducing yield loss by salt stress. Here, we used a novel plant growth regulator B2, which is a functional analogue of ABA. With the aim to determine whether B2 alleviates salt stress on maize, we studied its function under hydroponic conditions. When the second leaf was fully developed, it was pretreated with 100 µM ABA, 0.01 µM B2, 0.1 µM B2, and 1 µM B2, independently. After 5 days treatment, NaCl was added into the nutrient solution for salt stress. Our results showed that B2 could enhance salt tolerance in maize, especially when the concentration was 1.0 µMol·L-1. Exogenous application of B2 significantly enhanced root growth, and the root/shoot ratio increased by 7.6% after 6 days treatment under salt stress. Compared with control, the ABA level also decreased by 31% after 6 days, which might have resulted in the root development. What is more, B2 maintained higher photosynthetic capacity in maize leaves under salt stress conditions and increased the activity of antioxidant enzymes and decreased the generation rate of reactive oxygen species by 16.48%. On the other hand, B2 can enhance its water absorption ability by increasing the expression of aquaporin genes ZmPIP1-1 and ZmPIP1-5. In conclusion, the novel plant growth regulator B2 can effectively improve the salt tolerance in maize.

Exploring the role of Aquaporins (AQPs) in LPS induced systemic inflammation and the ameliorative effect of Garcinia in male Wistar rat.

The Aquaporins (AQPs) could prove to be striking targets of inflammation. The aim of this study was to study the involvement of AQPs and explore the anti-inflammatory activity of Garcinia extract in LPS induced acute systemic inflammation in Wistar rats. Adult male Wistar rats (n = 6) were pretreated with Garcinia orally twice for 7 days, followed by a single intraperitoneal dose (5.5 mg/kgbw) of LPS. Serum ALT, AST, ALP, Creatinine, Urea and BUN, nitric oxide, prostaglandin, cytokine and chemokine levels were measured. LC-MS analysis of Garcinia was performed to identify the phytoconstituents present. The iNOS and COX enzyme activity were determined in the target tissues. qPCR analysis of inos, cox-2 and aqps was performed. Relative protein expression of AQPs was studied by Western blot analysis. Molecular docking studies were performed to study the interaction of garcinol and hydroxycitric acid, the two important phytoconstituents of Garcinia with AQP. The qPCR analysis showed tissue-specific up-regulation of aqp1, aqp3, aqp4 and aqp8 in LPS induced rats. Garcinia extract treatment effectively lowered the mRNA expression of these AQPs. Garcinia extract significantly inhibited the LPS-induced NO, prostaglandin, cytokine and chemokine production in serum and also decreased tissue-specific transcript level of inos and cox-2, thus suggesting the anti-inflammatory role of Garcinia. Also, docking studies revealed interactions of garcinol and hydroxycitric acid with AQP1, 3, 4 and 8. Therefore, the present study suggests the possible involvement of AQP1, 3, 4 and 8 in inflammation and the efficacy of Garcinia extract as an anti-inflammatory agent. Therefore, AQPs can act as prognostic markers of inflammation and can be targeted with Garcinia extract.

Uterine aquaporin expression is dynamically regulated by estradiol and progesterone and ovarian stimulation disrupts embryo implantation without affecting luminal closure.

The study investigated the effect of normal and supraphysiological (resulting from gonadotropin-dependent ovarian stimulation) levels of estradiol (E2) and progesterone (P4) on mouse uterine aquaporin gene/protein (Aqp/AQP) expression on Day 1 (D1) and D4 of pregnancy. The study also examined the effect of ovarian stimulation on uterine luminal closure and uterine receptivity on D4 of pregnancy and embryo implantation on D5 and D7 of pregnancy. These analyses revealed that the expression of Aqp3, Aqp4, Aqp5 and Aqp8 is induced by E2 while the expression of Aqp1 and Aqp11 is induced by P4. Additionally, P4 inhibits E2 induction of Aqp3 and Aqp4 expression while E2 inhibits Aqp1 and Aqp11 expression. Aqp9, however, is constitutively expressed. Ovarian stimulation disrupts Aqp3, Aqp5 and Aqp8 expression on D4 and AQP1, AQP3 and AQP5 spatial expression on both D1 and D4, strikingly so in the myometrium. Interestingly, while ovarian stimulation has no overt effect on luminal closure and uterine receptivity, it reduces implantation events, likely through a disruption in myometrial activity and embryo development. The wider implication of this study is that ovarian stimulation, which results in supraphysiological levels of E2 and P4 and changes (depending on the degree of stimulation) in the E2:P4 ratio, triggers abnormal expression of uterine AQP during pregnancy, and this is associated with implantation failure. These findings lead us to recognize that abnormal expression would also occur under any pathological state (such as endometriosis) that is associated with changes in the normal E2:P4 ratio. Thus, infertility among these patients might in part be linked to abnormal uterine AQP expression.

Affinity tag effect on the salt rejection potential of Halomonas elongata aquaporin incorporated in thin film nanocomposite membrane.

In this study, effect of affinity tags, Histidine (His) and Glutathione-S-Transferase (GST), on the activity of halophilic aquaporin was analyzed. The gene coding for H. elongata aquaporin was cloned into pET28a vector and expressed in E. coli BL21 successfully. Stopped flow light scattering measurements showed that His-tagged aquaporin is functional. The difference in the filtration parameters caused by affinity tags were determined by using thin film composite nano-filtration (NFC) membranes prepared with the aquaporins. At 100 mM salt concentration, water permeability (L/m2.h) and the % salt rejection of NFC membranes produced with the His-tagged aquaporin was found to be higher than that of the membrane with GST-tagged aquaporin. Salt rejection of His-tagged aquaporin-membrane was found to be 53% with a lower solute permeability value (B). Use of short affinity tag (His tag) for cloning resulted in higher solute rejection ability of TFC membranes prepared with H. elongata aquaporins.

miR-27a-3p protects against blood-brain barrier disruption and brain injury after intracerebral hemorrhage by targeting endothelial aquaporin-11.

Previous studies have reported that miR-27a-3p is down-regulated in the serum of patients with intracerebral hemorrhage (ICH), but the implication of miR-27a-3p down-regulation in post-ICH complications remains elusive. Here we verified miR-27a-3p levels in the serum of ICH patients by real-time PCR and observed that miR-27a-3p is also significantly reduced in the serum of these patients. We then further investigated the effect of miR-27a-3p on post-ICH complications by intraventricular administration of a miR-27a-3p mimic in rats with collagenase-induced ICH. We found that the hemorrhage markedly reduced miR-27a-3p levels in the hematoma, perihematomal tissue, and serum and that intracerebroventricular administration of the miR-27a-3p mimic alleviated behavioral deficits 24 h after ICH. Moreover, ICH-induced brain edema, vascular leakage, and leukocyte infiltration were also attenuated by this mimic. Of note, miR-27a-3p mimic treatment also inhibited neuronal apoptosis and microglia activation in the perihematomal zone. We further observed that the miR-27a-3p mimic suppressed the up-regulation of aquaporin-11 (AQP11) in the perihematomal area and in rat brain microvascular endothelial cells (BMECs). Moreover, miR-27a-3p down-regulation increased BMEC monolayer permeability and impaired BMEC proliferation and migration. In conclusion, miR-27a-3p down-regulation contributes to brain edema, blood-brain barrier disruption, neuron loss, and neurological deficits following ICH. We conclude that application of exogenous miR-27a-3p may protect against post-ICH complications by targeting AQP11 in the capillary endothelial cells of the brain.

Regulation mechanism of aquaporin 9 gene on inflammatory response and cardiac function in rats with myocardial infarction through extracellular signal-regulated kinase1/2 pathway.

The aim of this study was to investigate the regulation mechanism of aquaporin 9 (AQP9) gene on inflammatory response and cardiac function in rats with myocardial infarction (MI) through extracellular signal-regulated kinase1/2 (ERK1/2) pathway. The constructed rats models of MI were randomly divided into 6 groups: control group (sham operation group, MI modeling sham operation), model group (MI modeling), NC group (MI modeling, tail vein injection of AQP9 negative control sequence vector), AQP9 shRNA group (MI modeling, tail vein injection of AQP9 shRNA plasmid vector), U0126 group (MI modeling, tail vein injection of ERK signaling pathway inhibitor), and AQP9 shRNA + U0126 group. The hemodynamics and cardiac function of rats in each group were detected on the seventh day of modeling. The levels of AQP9 and inflammatory factors [tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10)] in peripheral blood of rats were detected by ELISA method. qRT-PCR and western blot were used to detect the mRNA and protein expression of AQP9, ERK1/2, B-cell lymphoma-2 (Bcl-2), Bcl-associated x (Bax) in the myocardial tissue of rats. TTC and TUNEL staining were used to observe myocardial infarct size and apoptosis of myocardial cells in each group. Compared with control group, the levels of heart rate, left ventricular end-diastolic pressure, TNF-α, and IL-6 were increased in each group of rats with MI (all p < 0.05), while the levels of systolic blood pressure, diastolic blood pressure, mean arterial pressure, left ventricular systolic pressure, and IL-10 were significantly decreased (all p < 0.05). The mRNA and protein expression levels of AQP9, ERK1/2 phosphorylation and Bax were significantly increased, as well as the myocardial infarct size, apoptosis index of myocardial tissue (all p < 0.05), the mRNA and protein expression levels of Bcl-2 were significantly decreased (all p < 0.05). The AQP9 gene knock-down or exogenous administration of the ERK1/2 inhibitor U0126 could improve the above indexes. However, the combination of AQP9 gene knock-down and U0126 showed no further effect. Silencing AQP9 gene can inhibit the activation of ERK1/2 signaling pathway, attenuate the inflammatory response in rats with MI, inhibit apoptosis of myocardial cells, and improve cardiac function.

A trans-acting Variant within the Transcription Factor RIM101 Interacts with Genetic Background to Determine its Regulatory Capacity.

Most genetic variants associated with disease occur within regulatory regions of the genome, underscoring the importance of defining the mechanisms underlying differences in regulation of gene expression between individuals. We discovered a pair of co-regulated, divergently oriented transcripts, AQY2 and ncFRE6, that are expressed in one strain of Saccharomyces cerevisiae, ∑1278b, but not in another, S288c. By combining classical genetics techniques with high-throughput sequencing, we identified a trans-acting single nucleotide polymorphism within the transcription factor RIM101 that causes the background-dependent expression of both transcripts. Subsequent RNA-seq experiments revealed that RIM101 regulates many more targets in S288c than in ∑1278b and that deletion of RIM101 in both backgrounds abrogates the majority of differential expression between the strains. Strikingly, only three transcripts undergo a significant change in expression after swapping RIM101 alleles between backgrounds, implying that the differences in the RIM101 allele lead to a remarkably focused transcriptional response. However, hundreds of RIM101-dependent targets undergo a subtle but consistent shift in expression in the S288c RIM101-swapped strain, but not its ∑1278b counterpart. We conclude that ∑1278b may harbor a variant(s) that buffers against widespread transcriptional dysregulation upon introduction of a non-native RIM101 allele, emphasizing the importance of accounting for genetic background when assessing the impact of a regulatory variant.

Endogenous bioelectric currents promote differentiation of the mammalian lens.

The functional roles of bioelectrical signals (ES) created by the flow of specific ions at the mammalian lens equator are poorly understood. We detected that mature, denucleated lens fibers expressed high levels of the α1 and ß1 subunits of Na+ /K+ -ATPase (ATP1A1 and ATP1B1 of the sodium pump) and had a hyperpolarized membrane potential difference (Vmem ). In contrast, differentiating, nucleated lens fiber cells had little ATP1A1 and ATP1B1 and a depolarized Vmem . Mimicking the natural equatorial ES with an applied electrical field (EF) induced a striking reorientation of lens epithelial cells to lie perpendicular to the direction of the EF. An EF also promoted the expression of ß-crystallin, aquaporin-0 (AQP0) and the Beaded Filament Structural Protein 2 (BFSP2) in lens epithelial cells (LECs), all of which are hallmarks of differentiation. In addition, applied EF activated the AKT and CDC2 and inhibition of AKT reduced the activation of CDC2. Our results indicate that the endogenous bioelectrical signal at the lens equator promotes differentiation of LECs into denucleated lens fiber cells via depolarization of Vmem. Development of methods and devices of EF application or amplification in vivo may supply a novel treatment for lens diseases and even promote regeneration of a complete new lens following cataract surgery.

Similar Aquaporin9 and MAPK Expression Profiles in the Liver of Types 1 and 2 Diabetes Mellitus.

Aquaporin-9 (AQP9) is an aquaglyceroporin that biophysically conducts water, glycerol, and other small solutes. AQP9 is expressed in hepatocytes on the sinusoidal surfaces of hepatocyte plates in the liver, where it is considered responsible for the glycerol uptake in gluconeogenesis. However, limited information is available on the expression and regulating mechanism of AQP9 in different hyperglycemia models. Thus, this study examined the expression patterns of AQP9 and mitogen-activated protein kinase (MAPK) in Types 1 and 2 diabetes mellitus (DM) to clarify the roles and regulating mechanism of AQP9 in gluconeogenesis. Compared with the control group, the AQP9 expression significantly increased in both Types 1 and 2 DM, and the increased expression was associated with the activation of phosphorylated JNK (p-JNK) and the inhibition of phosphorylated p38 (p-p38). By contrast, phosphorylated ERK remained stable in the liver with Type 1 or 2 DM. These effects could be reversed by insulin treatment. That is, insulin downregulated AQP9 by inhibiting p-JNK and activating p-p38. The upregulation of AQP9 could be involved in gluconeogenesis and co-regulated by the JNK and p38 MAPK pathway in both Types 1 and 2 DM.

From channel proteins to industrial biomimetic membrane technology.

Biomimetic membrane technology, based on the use of nano-scale functional additives in the form of channel proteins or artificially made channel structures, represents an attractive way of optimizing membrane separation technology. However, the nano-scale nature of the additives inherently points to the challenge in up-scaling the membranes to square meter areas. Thus, the ability to up-scale the processes involved in manufacturing will be crucial for translating the protein/nano-science into technology. Here we discuss how highly selective aquaporin proteins can be used to enhance the performance of the classical thin film composite membrane, and how this can be used in relevant membrane elements and module form factors. A particular up-scaling challenge lies in securing large scale membrane protein production. We demonstrate our framework for making batch amounts which are compatible with the large scale production of biomimetic membranes for water purification based on the use of the E. coli expression system.

Expression of AQP2, AQP4 and AQP 8 in mouse intestine induced by unprocessed and processed Euphorbia lathyris.

The present research was designed to study expression of AQP2, AQP4 and AQP8 in mouse intestines induced by unprocessed and processed Euphorbia lathyris. KM mice were given by different dose lavage of unprocessed and processed Euphorbia lathyris, Euphorbia factor L1, Euphorbia factor L2, Euphorbia factor L3. Samples of mouse intestine were collected for protein levels of AQP2, AQP 4 and AQP 8 which were assessed by immunohistochemical staining and mRNA expression of AQP2, AQP 4 and AQP 8 which were quantified by Real Time-PCR. Comparing to the normal control group, the protein levels of AQP2, AQP 4 and AQP 8 were significantly decreased (P<0.05)by Semen Euphorbiae group and Semen Euphorbiae Pulveratum group (unprocessed and processed Euphorbia lathyris) induced. Protein expression of AQP2, AQP 4 and AQP 8 in the Euphorbia factor L1, Euphorbia factor L2 and Euphorbia factor L3 group were not significantly lower than normal control group. There had no differences on the levels of AQP2 and AQP 8 mRNA expressions between the high-dose group of semen Euphorbiae group, semen Euphorbiae Pulveratum group and positive control group, while significantly lower than normal control group (P<0.05). Expression of AQP4 mRNA in the Semen Euphorbiae group and Semen Euphorbiae Pulveratum group has not significantly decreased. But levels of AQP2, AQP 4 and AQP 8 mRNA in the Euphorbia factor L1 group had no significant differences in normal control group and positive control group. These findings suggest that semen Euphorbiae could regulate expression of AQP2, AQP 4 and AQP 8 protein and mRNA, which may be the possible one reason of semen Euphorbiae induces diarrhea. The semen Euphorbiae group has more significant effects on the levels of AQP2, AQP 4 and AQP 8 protein and mRNA than semen Euphorbiae Pulveratum group, which may be one of the mechanisms of processing attenuation.

The expression patterns of aquaporin 9, vacuolar H+-ATPase, and cytokeratin 5 in the epididymis of the common vampire bat.

Desmodus rotundus is a vampire bat species that inhabits Latin America. Some basic aspects of this species' biology are still unknown, as the histophysiological characteristics of the male reproductive tract. Our study has focused on its epididymis, which is an important organ for performing a variety of functions, especially the sperm maturation and storage. The aim of this study was to identify principal, narrow, clear, and basal cells using cell-specific markers such as aquaporin 9 (AQP9), vacuolar H+-ATPase (V-ATPase), and cytokeratin 5 (KRT5). Principal cells were labeled by AQP9 from initial segment to cauda region in their stereocilia. They were shown with a columnar shape, whereas V-ATPase-rich cells were identified with a goblet-shaped body along the entire epididymis, including the initial segment, which were named as clear cells. Pencil-shaped V-ATPase-rich cells (narrow cells) were not detected in the initial segment of the bat epididymis, unlike in the rodent. Basal cells were labeled by KRT5 and were located at the basal portion of the epithelium forming a dense network. However, no basal cells with a luminal-reaching body extension were observed in the bat epididymis. In summary, epithelial cells were identified by their specific markers in the vampire bat epididymis. Principal and basal cells were labeled by AQP9 and KRT5, respectively. Narrow cells were not observed in the vampire bat epididymis, whereas clear cells were identified by V-ATPase labeling along the entire duct in a goblet-shaped body. In addition, no luminal-reaching basal cells were observed in the vampire bat epididymis.

A fusion protein of the synthetic IgG-binding domain and aequorin: Expression and purification from E. coli cells and its application.

Aequorin is a Ca2+-binding photoprotein that is a complex of apoaequorin (apoAQ) and 2-peroxycoelenterazine. In this study, the fusion protein (ZZ-apoAQ) composed of the synthetic IgG-binding domain (ZZ domain) derived from Staphylococcus aureus protein A and apoAQ was expressed into the periplasmic space of Escherichia coli cells. ZZ-apoAQ was highly purified using Ni-chelate affinity chromatography followed by IgG affinity chromatography. ZZ-AQ was prepared from purified ZZ-apoAQ by incubation with coelenterazine and was characterized, including its luminescence properties. ZZ-AQ could be used as a reporter for detecting IgG and the measurable range of IgG coated on a 96-well plate was 1-1000 ng/mL.

Is electrolyte transfer across the urothelium important?: ICI-RS 2015.

AIMS: This article summarizes discussion at the International Consultation on Incontinence Research Society (ICI-RS) 2015 meeting of urine modification in the urinary tract by the urothelium. It considers the literature and proposes pertinent questions that need to be addressed to understand this phenomenon within a physiological context. METHODS: Following the ICI-RS meeting, publications in PubMed relating to urine modification in the renal pelvis, ureter, and bladder were reviewed. RESULTS: Historically, the urothelium has been simply considered as a passive, impermeable barrier, preventing contact between urine and the underlying cells. In addition to the ability of the umbrella cells to modify the surface area of the urothelium during bladder filling, the urothelium may also be involved in modifying urine composition. Several lines of evidence support the hypothesis that electrolytes and water can be reabsorbed by the urothelium and that this may have physiological relevance. Firstly, urothelial cells express several types of aquaporins and ion channels; the membrane expression of which is modulated by the extracellular concentration of ions including Na+ . Secondly, studies of urine composition in the renal pelvis and bladder demonstrate urine modification, indicating that water and/or electrolyte transport has occurred. Thirdly, hibernating mammals, with urothelial and bladder wall histology similar to non-hibernating mammals are known to produce and reabsorb urine daily, during long periods of hibernation. CONCLUSIONS: The phenomenon of urine modification by the urothelium may be physiologically important during normal bladder filling. Research should be focused on investigating how this may change in conditions of urinary dysfunction.

Aquaporin in different moult stages of a freshwater decapod crustacean: Expression and participation in muscle hydration control.

Crustaceans, during their moult cycle, at the stages of both pre-moult and post-moult, need water uptake. This movement of water creates a challenge for the regulation of cell volume. The cells of freshwater decapods require a high regulatory capacity to deal with hyposmotic stresses, given the need to face dilution of the haemolymph during their moult cycles. This study investigated the variation in the expression of water channels (aquaporins) along the moult cycle of a freshwater palaemonid shrimp, focusing on their role in cell volume regulation. Moults in Palaemonetes argentinus have been investigated along three stages of its moult cycle: intermoult, late pre-moult and recent post-moult. For the evaluation of tissue volume regulation, the weight of isolatedmuscle, subjected to isosmotic and hyposmotic salines, was followed for 60min. The expression of AQP during the different moult stages was evaluated by immunocytochemistry. Muscle from the three moult stages in isosmotic conditions showed the same pattern of tissue volume regulation. When muscle from animals in pre-moult and intermoult were submitted to hyposmotic stress they swell, followed by volume regulation, while in post-moult the regulation is compromised. The difference in volume regulatory control between pre-moult and post-moult may be related to a possible regulation of water channels, as AQP expression was equal at these stages. This study presents novel findings for crustaceans in general, in the demonstration that AQP expression changes during the moult cycle of a decapod crustacean, together with the regulation of cell volume with the participation of AQPs.

Brattleboro rats have impaired apical membrane water permeability regulation in the outer medullary collecting duct principal cells.

Vasopressin (AVP) regulates the body salt-water balance. Brattleboro rats carry an AVP gene mutation resulting in a recessive form of central diabetes insipidus, being ideal for AVP deficiency studies. Herein, we studied the water permeability of the apical and basolateral sides of outer medullary collecting duct (OMCD) principal cells in response to dDAVP (a V2 receptor agonist) administration in Wistar and Brattleboro rats. Biophysical measurements of the water permeability (Pf ) of isolated OMCD principal cells were performed with the calcein quenching method with/without dDAVP (10-8  mol/L). mRNA transcripts and protein levels of AQP2, AQP3 and AQP4 were assessed by RT-PCR and western blot respectively. dDAVP increased the apical and basolateral Pf of OMCD principal cells in Wistar rats, while in Brattleboro rats this effect was present basolaterally. Long-term dDAVP administration in both strains resulted in a significant increase in mRNA expression of all assessed AQP's while only the protein levels of AQP2 and AQP3 were significantly increased. Short-term (20 minutes) dDAVP treatment of isolated OMCD fragments resulted in significantly increased plasma membrane expression of AQP2 in Wistar rats and of AQP2 and AQP3 in Brattleboro rats. In summary, dDAVP induces different expression of AQP2, AQP3 and AQP4 in Wistar and Brattleboro rats during short- and long-term treatment. In Wistar rats dDAVP mainly increased AQP2 expression while in Brattleboro rats it increased functional water permeability mainly by AQP3 expression.

Specific aquaporins facilitate Nox-produced hydrogen peroxide transport through plasma membrane in leukaemia cells.

In the last decade, the generation and the role of reactive oxygen species (ROS), particularly hydrogen peroxide, in cell signalling transduction pathways have been intensively studied, and it is now clear that an increase of ROS level affects cellular growth and proliferation pathways related to cancer development. Hydrogen peroxide (H2O2) has been long thought to permeate biological membranes by simple diffusion since recent evidence challenged this notion disclosing the role of aquaporin water channels (AQP) in mediating H2O2 transport across plasma membranes. We previously demonstrated that NAD(P)H oxidase (Nox)-generated ROS sustain glucose uptake and cellular proliferation in leukaemia cells. The aim of this study was to assess whether specific AQP isoforms can channel Nox-produced H2O2 across the plasma membrane of leukaemia cells affecting downstream pathways linked to cell proliferation. In this work, we demonstrate that AQP inhibition caused a decrease in intracellular ROS accumulation in leukaemia cells both when H2O2 was produced by Nox enzymes and when it was exogenously added. Furthermore, AQP8 overexpression or silencing resulted to modulate VEGF capacity of triggering an H2O2 intracellular level increase or decrease, respectively. Finally, we report that AQP8 is capable of increasing H2O2-induced phosphorylation of both PI3K and p38 MAPK and that AQP8 expression affected positively cell proliferation. Taken together, the results here reported indicate that AQP8 is able to modulate H2O2 transport through the plasma membrane affecting redox signalling linked to leukaemia cell proliferation.

Aquaporins in developing rice grains.

During rice grain filling, grain moisture content and weight show dynamic changes. We focused on the expression of all 33 rice aquaporins in developing grains. Only two aquaporin genes, OsPIP2;1 and OsTIP3;1, were highly expressed in the period 10-25 days after heading (DAH). High-temperature treatment from 7 to 21 DAH abolished the dynamic up-regulation of OsPIP2;1 in the period 15-20 DAH, whereas OsTIP3;1 expression was not affected. Immunohistochemical analysis revealed that OsPIP2;1 was present in the starchy endosperm, nucellar projection, nucellar epidermis, and dorsal vascular bundles, but not in the aleurone layer. OsTIP3;1 was present in the aleurone layer and starchy endosperm. Water transport activity of recombinant OsTIP3;1 was low, in contrast to the high activity of recombinant OsPIP2;1 we reported previously. Our data suggest that OsPIP2;1 and OsTIP3;1 have distinct roles in developing grains.