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Pond management with chemical and biological agents that reduce overgrowth of algae is an important means of maintaining water quality in residential ponds, yet the effects on nontarget species are not fully understood. We assessed the impact of Aquashade (a common nontoxic pond dye) and copper sulfate (a toxic algaecide) on American toad (Anaxyrus americanus), northern leopard frog (Lithobates pipiens), and Cope's gray treefrog (Hyla chrysoscelis) metamorphosis in outdoor mesocosm experiments. We also evaluated the relative impact of tadpole grazing versus chemical treatment on phytoplankton and periphyton abundance. We found no significant effects of pond management treatment on anuran metamorphosis, suggesting that addition of Aquashade and copper sulfate at tested concentrations does not significantly impact anurans under these experimental conditions. Interestingly, we found that the presence of tadpoles more strongly reduced algal abundance than Aquashade or copper sulfate by significantly decreasing phytoplankton and periphyton abundance over time. The present study suggests that anuran tadpoles may be effective at maintaining water quality, and that Aquashade and copper sulfate may have minimal effects on amphibian metamorphosis. Environ Toxicol Chem 2023;42:213-224. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Arginine vasopressin (AVP) stimulates the concentration of renal urine by increasing the principal cell expression of aquaporin-2 (AQP2) water channels. Prostaglandin E2 (PGE2) and prostaglandin2α (PGF2α) increase the water absorption of the principal cell without AVP, but PGE2 decreases it in the presence of AVP. The underlying mechanism of this paradoxical response was investigated here. Mouse cortical collecting duct (mkpCCDc14) cells mimic principal cells as they endogenously express AQP2 in response to AVP. PGE2 increased AQP2 abundance without desmopressin (dDAVP), while in the presence of dDAVP, PGE2, and PGF2α reduced AQP2 abundance. dDAVP increased the cellular PGD2 and PGE2 release and decreased the PGF2α release. MpkCCD cells expressed mRNAs for the receptors of PGE2 (EP1/EP4), PGF2 (FP), and TxB2 (TP). Incubation with dDAVP increased the expression of EP1 and FP but decreased the expression of EP4. In the absence of dDAVP, incubation of mpkCCD cells with an EP4, but not EP1/3, agonist increased AQP2 abundance, and the PGE2-induced increase in AQP2 was blocked with an EP4 antagonist. Moreover, in the presence of dDAVP, an EP1/3, but not EP4, agonist decreased the AQP2 abundance, and the addition of EP1 antagonists prevented the PGE2-mediated downregulation of AQP2. Our study shows that in mpkCCDc14 cells, reduced EP4 receptor and increased EP1/FP receptor expression by dDAVP explains the differential effects of PGE2 and PGF2α on AQP2 abundance with or without dDAVP. As the V2R and EP4 receptor, but not the EP1 and FP receptor, can couple to Gs and stimulate the cyclic adenosine monophosphate (cAMP) pathway, our data support a view that cells can desensitize themselves for receptors activating the same pathway and sensitize themselves for receptors of alternative pathways.

Residential areas are increasing on the landscape but their ability to provide suitable habitat is often based on management for recreational use and aesthetics. Amphibians rely on both aquatic and terrestrial habitat making them susceptible to changes in land-cover and land-use. As anthropogenic land-use change increases, it is imperative to assess how pond management practices impact aquatic communities. We assessed the impact of Aquashade (a common non-toxic pond dye) and copper sulfate (a toxic algaecide) on American toad (Anaxyrus americanus), northern leopard frog (Lithobates pipiens), and Cope’s gray treefrog (Hyla chrysoscelis) metamorphosis in outdoor mesocosm experiments. We also evaluated the relative impact of tadpole grazing and chemical treatment on phytoplankton and periphyton abundance. We found no significant effects of pond management treatment on anuran metamorphosis, suggesting that addition of Aquashade and copper sulfate does not significantly impact anurans under these experimental conditions. However, while we found no differences in phytoplankton and periphyton abundance due to pond management treatment, presence of tadpoles significantly decreased phytoplankton and periphyton abundance over time. This result suggests that the creation of suitable pond habitat for anuran tadpoles may be an efficient and ecologically beneficial form of pond management treatment to maintain water quality.

Contains fulltext : 97303.pdf (Publisher’s version ) (Closed access) Vasopressin-induced water reabsorption coincides with phosphorylation of aquaporin-2 (AQP2) at S256 (pS256), dephosphorylation at S261, and its translocation to the apical membrane, whereas treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA) induces AQP2 ubiquitination at K270, its internalization, and lysosomal degradation. In this study we investigated the relationship between S256 and S261 phosphorylation in AQP2 and its ubiquitination and trafficking in MDCK cells. Forskolin stimulation associated with increased pS256 and decreased pS261 AQP2, indicating that MDCK cells are a good model. After forskolin stimulation, TPA-induced ubiquitination of AQP2 preceded phosphorylation of AQP2 at S261, which in the first instance occurred predominantly on ubiquitinated AQP2. Forskolin-induced changes in pS261 were also observed for AQP2-S256A and AQP2-S256D, which constitutively localize in vesicles and the apical membrane, respectively. Although pS261 varies with forskolin as with wild-type AQP2, AQP2-S256A is not increased in its ubiquitination. Our data reveal that pS261 occurred independently of AQP2 localization and suggest that pS261 follows ubiquitination and endocytosis and may stabilize AQP2 ubiquitination and intracellular localization. The absence of increased ubiquitination of AQP2-S256A indicates that its intracellular location is due to the lack of pS256. Furthermore, AQP2-S261A and AQP2-S261D localized to vesicles, which was due to their increased ubiquitination, because changing K270 into Arg in both mutants resulted in their localization in the apical membrane. Although still increased in its ubiquitination, AQP2-S256D-S261D localized in the apical membrane. AQP2-S256D-K270R-Ub, however, localized to intracellular vesicles. Although our localization of AQP2-S261A/D is different from that of others, these data indicate that constitutive S256 phosphorylation counterbalances S261D-induced ubiquitination and internalization or changes its structure to allow distribution to the apical membrane. The vesicular localization of AQP2-S256D-K270R-Ub, however, indicates that the dominant apical sorting of S256D can again be overruled by constitutive ubiquitination. These data indicate that the membrane localization of AQP2 is determined by the balance of the extents of phosphorylation and ubiquitination.

Contains fulltext : 95770.pdf (Publisher’s version ) (Closed access) Water homeostasis is regulated by a wide variety of hormones. When in need for water conservation, vasopressin, released from the brain, binds renal principal cells and initiates a signaling cascade resulting in the insertion of aquaporin-2 (AQP2) water channels in the apical membrane and water reabsorption. Conversely, hormones, including extracellular purines and dopamine, antagonize AVP-induced water permeability, but their mechanism of action is largely unknown, which was investigated here. Addition of these hormones to mpkCCD cells decreased total and plasma membrane abundance of AVP-induced AQP2, partly by increasing its internalization to vesicles and lysosomal degradation. This internalization was ubiquitin dependent, because the hormones increased AQP2 ubiquitination, and the plasma membrane localization of AQP2-K270R, which cannot be monoubiquitinated, was unaffected by these hormones. Both hormones also increased AQP2 phosphorylation at S261, which followed ubiquitination, but was not essential for hormone-induced AQP2 degradation. A similar process occurs in vivo, as incubation of dDAVP-treated kidney slices with both hormones also resulted in the internalization and S261 phosphorylation of AQP2. Both hormones also reduced cAMP and AQP2 mRNA levels, suggesting an additional effect on AQP2 gene transcription. Interestingly, phorbol esters only reduced AQP2 through the first pathway. Together, our results indicate that ATP and dopamine counteract AVP-induced water permeability by increasing AQP2 degradation in lysosomes, preceded by ubiquitin-dependent internalization, and by decreasing AQP2 gene transcription by reducing the AVP-induced cAMP levels.

Contains fulltext : 88744.pdf (Publisher’s version ) (Closed access) BACKGROUND: Arginine vasopressin (AVP) binding to the V2 receptor (V2R) in renal collecting duct principal cells induces a cAMP signalling cascade resulting in the activation of protein kinase A (PKA), translocation of aquaporin-2 (AQP2) to the apical membrane and an increase in AQP2 expression. Consequently, concentration of urine is initiated. X-linked nephrogenic diabetes insipidus (NDI), characterized by the inability to concentrate urine in response to AVP, is caused by mutations in the V2R gene. Initiation of AQP2 translocation, while circumventing the V2R-cAMP-PKA pathway has been suggested as a putative therapy for these patients. In this respect, the activation of a cAMP-independent and cGMP-dependent pathway for AQP2 membrane insertion by different cyclic guanosine monophosphate (cGMP) pathway activators, such as atrial natriuretic peptide (ANP), l-arginine and 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), has been put forward. However, it is unclear whether they can increase AQP2 expression. METHODS: Mouse cortical collecting duct (mpkCCD) cells were incubated with ANP, l-arginine and 8-Br-cGMP for 2 h and subjected to immunocytochemistry and cell surface biotinylation assays to examine their effect on AQP2 translocation. To test the effect of cGMP pathway activators on AQP2 expression, the mpkCCD cells were treated with dDAVP, ANP and l-arginine for 4 days, or with 8-Br-cGMP for the last day. AQP2 protein levels were determined by immunoblotting. RESULTS: ANP, l-arginine and 8-Br-cGMP induced the translocation of AQP2 in the mpkCCD cells. However, in contrast to dDAVP, ANP, l-arginine and 8-Br-cGMP did not increase the expression of AQP2. CONCLUSIONS: Our results suggest that while activators of the cGMP pathway are likely beneficial in the treatment of X-linked NDI, their ability to relieve NDI in the patients may be improved when combined with agents stimulating AQP2 expression. 01 januari 2010

Contains fulltext : 81345.pdf (Publisher’s version ) (Open Access) Vasopressin regulates water reabsorption in the collecting duct, but extracellular nucleotides modulate this regulation through incompletely understood mechanisms. We investigated these mechanisms using immortalized mouse collecting duct (mpkCCD) cells. Basolateral exposure to dDAVP induced AQP2 localization to the apical membrane, but co-treatment with ATP internalized AQP2. Because plasma membrane-bound P2 receptors (P2R) mediate the effects of extracellular nucleotides, we examined the abundance and localization of P2R in mpkCCD cells. In the absence of dDAVP, P2Y(1) and P2Y(4) receptors localized to the apical membrane, whereas P2X(2), P2X(4), P2X(5), P2X(7), P2Y(2), P2Y(11), and P2Y(12) receptors localized to the cytoplasm. dDAVP induced gene expression of P2X(1), which localized to the apical domain, and led to translocation of P2X(2) and P2Y(2) to the apical and basolateral membranes, respectively. In co-expression experiments, P2R activation decreased membrane AQP2 and AQP2-mediated water permeability in Xenopus oocytes expressing P2X(2), P2Y(2,) or P2Y(4) receptors, but not in oocytes expressing other P2R subtypes. In summary, these data suggest that AQP2-mediated water transport is downregulated not only by basolateral nucleotides, mediated by P2Y(2) receptors, but also by luminal nucleotides, mediated by P2X(2) and/or P2Y(4) receptors.

Aquaporins (AQPs) are central players in mammalian physiology, allowing efficient water transport through cellular membranes. To date, 13 different aquaporins have been identified in mammals (AQP0-AQP12). Knocking out genes in mice and identification of mutations in the human genes provided important information on the role of AQPs in normal physiology. While the physiological role of many AQPs only becomes clear when the putative function is challenged, the lack of AQP2 directly results in a disease phenotype. Aquaporin 2 is highly expressed in the principal cells of the renal collecting duct, where it shuttles between intracellular storage vesicles and the apical membrane. Upon hypernatraemia or hypovolaemia, the antidiuretic hormone vasopressin (AVP) is released from the pituitary into blood and binds to its type 2 receptor on renal principal cells. This initiates a cAMP signalling cascade resulting in the translocation of AQP2-bearing vesicles to the apical membrane. Subsequently, pro-urinary water reabsorption and urine concentration occurs. This process is reversed by a reduction in circulating AVP levels, which is obtained with the establishment of isotonicity. In humans, mutations in the AQP2 gene cause congenital nephrogenic diabetes insipidus (NDI), a disorder characterized by an inability to concentrate urine in response to vasopressin. Until the recent development of several congenital NDI mouse models, our knowledge on AQP2 regulation was primarily based on in vitro studies. This review focuses on the similarities between the in vitro and in vivo studies and discusses new insights into congenital NDI obtained from the mouse models.

Contains fulltext : 76117.pdf (Publisher’s version ) (Open Access) Vasopressin binding to the V2 receptor in renal principal cells leads to activation of protein kinase A, phosphorylation of aquaporin 2 (AQP2) at Ser256, and the translocation of AQP2 to the apical membrane, resulting in concentration of the urine. In contrast, phorbol ester-induced activation of protein kinase C pathway leads to ubiquitination of AQP2 at Lys270 and its internalization to multivesicular bodies, where it is targeted for lysosomal degradation or stored for recycling. Because little is known about the regulation of AQP2 trafficking, we used the carboxy-terminal tail of constitutively nonphosphorylated AQP2 (S256A) as a bait for interacting proteins in a yeast two-hybrid assay. We isolated lysosomal trafficking regulator-interacting protein 5 (LIP5) and found that LIP5 interacted with the proximal carboxy-terminal tail (L230-D243) of AQP2 in vitro but not with AQP3 or AQP4, which are also expressed in principal cells. Immunohistochemistry revealed that LIP5 co-localized with AQP2 in principal cells. LIP5 binding occurred independent of the state of Ser256 phosphorylation or Lys270 ubiquitination. LIP5 has been shown to facilitate degradation of the EGF receptor; here, LIP5 seemed to bind this receptor. Knockdown of LIP5 in mouse renal cells (mpkCCD) reduced the phorbol ester-induced degradation of AQP2 approximately two-fold. In summary, LIP5 binds cargo proteins and, considering the role of LIP5 in protein sorting to multivesicular bodies, plays a role in the degradation of AQP2, possibly by reducing the formation of late endosomes.

Concern about the impact of narrative worlds and their heroes offered by the media prompted research on encounters with moral models in experiential, narrative curricula. Researchers tracked the extension of a mandated Language Arts curriculum on “heroes” through the experiential narratives of four local heroes chosen collaboratively by teacher, students and researcher. They also elicited and analyzed responses from students to these narrative presentations in order to explore how students understood the narrative worlds presented to them. Instead of focusing on the personalities of the speakers, the researchers considered the experiential stories, and the moments of narrative encounter they offered, as the sources of immediate moral impact. However, this impact, it is suggested, did not adhere to a particular narrative in an undifferentiated manner. Instead, effects varied according to what a particular student brought to the encounter and how he or she was able to experience it. Material from tw...

Contains fulltext : 71463.pdf (Publisher’s version ) (Closed access) To prevent dehydration, terrestrial animals and humans have developed a sensitive and versatile system to maintain their water homeostasis. In states of hypernatremia or hypovolemia, the antidiuretic hormone vasopressin (AVP) is released from the pituitary and binds its type-2 receptor in renal principal cells. This triggers an intracellular cAMP signaling cascade, which phosphorylates aquaporin-2 (AQP2) and targets the channel to the apical plasma membrane. Driven by an osmotic gradient, pro-urinary water then passes the membrane through AQP2 and leaves the cell on the basolateral side via AQP3 and AQP4 water channels. When water homeostasis is restored, AVP levels decline, and AQP2 is internalized from the plasma membrane, leaving the plasma membrane watertight again. The action of AVP is counterbalanced by several hormones like prostaglandin E2, bradykinin, dopamine, endothelin-1, acetylcholine, epidermal growth factor, and purines. Moreover, AQP2 is strongly involved in the pathophysiology of disorders characterized by renal concentrating defects, as well as conditions associated with severe water retention. This review focuses on our recent increase in understanding of the molecular mechanisms underlying AVP-regulated renal water transport in both health and disease.

To regulate mammalian water homeostasis, arginine-vasopressin (AVP) induces phosphorylation and thereby redistribution of renal aquaporin-2 (AQP2) water channels from vesicles to the apical membrane. Vice versa, AVP (or forskolin) removal and hormones activating PKC cause AQP2 internalization, but the mechanism is unknown. Here, we show that a fraction of AQP2 is modified with two to three ubiquitin moieties in vitro and in vivo . Mutagenesis revealed that AQP2 is ubiquitinated with one K63-linked chain at K270 only. In Madin–Darby canine kidney cells, AQP2 ubiquitination occurs preferentially when present in the apical membrane, is transiently increased with forskolin removal or PKC activation, and precedes its internalization. Internalization kinetics assays with wild type (wt) and ubiquitination-deficient (K270R) AQP2 revealed that ubiquitination enhances AQP2 endocytosis. Electron microscopy showed that a translational fusion of AQP2 with ubiquitin (AQP2-Ub) localized particularly to internal vesicles of multivesicular bodies (MVBs), whereas AQP2-K270R largely localized to the apical membrane, early endosomes, and the limiting membrane of MVBs. Consistent with this distribution pattern, lysosomal degradation was extensive for AQP2-Ub, low for AQP2-K270R, and intermediate for wt-AQP2. Our data show that short-chain ubiquitination is involved in the regulated endocytosis, MVB sorting, and degradation of AQP2 and may be the mechanism used by AVP removal and PKC-activating hormones to reduce renal water reabsorption. Moreover, because several other channels are also (short-chain) ubiquitinated, our data suggest that ubiquitination may be a general mediator for the regulated endocytosis and degradation of channels in higher eukaryotes.

That processes of interpretation are unavoidable in any educational experience has not received serious attention in teaching and teacher education. One of the authors devised an exercise for her graduate class to help them understand practical aspects of hermeneutic theory in connection with rationality, truth and the nature of teaching and learning. The three co‐authors, each from a different cultural background, monitored and described their experiences of this exercise in order to explore the role of interpretation in teaching and learning. The students' interpretive activity was found to link subject matter to their past experience, reconstructing both. This process is described in detail and it is suggested that curricular spaces for interpretation be deliberately incorporated into teaching and learning situations. Le fait que des processus d'interpretation sont inevitables dans des experiences educationnelles n'a pas recu d'attention serieuse en enseignement ou en enseignement pedagogique. Un des a...

Two Canadians, both researchers, teachers and thesis writers, assume the voice of the other colleague in an arts‐based activity; we write letters that embody the voice of whiteness and the voice of a visible minority. By examining our experiences through arts‐based development strategies, we expand and transform our way of knowing the other and, in the process, enlarge our understanding of cultural self‐identity. We do so in the hopes of dialoguing issues of race and race relations in the field of multicultural education.

Contains fulltext : 87807.pdf (Publisher’s version ) (Closed access) Endocytosis, subsequent protein sorting into multivesicular bodies (MVBs), and eventual degradation in lysosomes compose an important mechanism for controlling protein expression on the plasma membrane. The lysosomal trafficking regulator interacting protein-5 (LIP5) is part of the complex protein machinery involved in MVB biosynthesis. LIP5 interacts with other players of the ESCRT machinery as well as with two known cargo proteins, AQP2 and EGFR, whose degradation is affected upon reduction of LIP5 expression. To investigate the expression and localization pattern of LIP5, we studied LIP5 protein expression in a mouse tissue panel and subjected various rodent and human tissues to immunohistochemistry. Immunoblotting revealed that, except for jejunum, LIP5 is expressed as a 42 kDa protein in all mouse tissues tested. Alternatively-spliced gene products could not be detected. Immunohistochemical studies revealed that in tissues positive for LIP5, LIP5 is detected in virtually all epithelial cells of the examined rodent and human tissues. The observed LIP5 expression in epithelial tissues suggests that LIP5 is of particular importance in the MVB sorting and degradation of proteins expressed in polarized cells. 01 februari 2010

Aquaporins (AQPs) are central players in mammalian physiology, allowing efficient water transport through cellular membranes. To date, 13 different aquaporins have been identified in mammals (AQP0-AQP12). Knocking out genes in mice and identification of mutations in the human genes provided important information on the role of AQPs in normal physiology. While the physiological role of many AQPs only becomes clear when the putative function is challenged, the lack of AQP2 directly results in a disease phenotype. Aquaporin 2 is highly expressed in the principal cells of the renal collecting duct, where it shuttles between intracellular storage vesicles and the apical membrane. Upon hypernatraemia or hypovolaemia, the antidiuretic hormone vasopressin (AVP) is released from the pituitary into blood and binds to its type 2 receptor on renal principal cells. This initiates a cAMP signalling cascade resulting in the translocation of AQP2-bearing vesicles to the apical membrane. Subsequently, pro-urinary water reabsorption and urine concentration occurs. This process is reversed by a reduction in circulating AVP levels, which is obtained with the establishment of isotonicity. In humans, mutations in the AQP2 gene cause congenital nephrogenic diabetes insipidus (NDI), a disorder characterized by an inability to concentrate urine in response to vasopressin. Until the recent development of several congenital NDI mouse models, our knowledge on AQP2 regulation was primarily based on in vitro studies. This review focuses on the similarities between the in vitro and in vivo studies and discusses new insights into congenital NDI obtained from the mouse models.

Item does not contain fulltext The von Hippel-Lindau (VHL) tumor suppressor gene regulates the extracellular matrix by controlling fibronectin deposition. To identify novel VHL target genes, we subjected mRNA from VHL-deficient RCC cells (786-0-pRC) and a transfectant re-expressing wildtype VHL (786-0-VHL) to differential expression profiling. Among the differentially expressed genes, we detected that fibronectin is upregulated in the presence of VHL, while it is not affected by hypoxia. Thus regulation of fibronectin deposition by VHL occurs at the transcriptional level, irrespective of oxygen levels.