Epitope-tagged Receptor Knock-in Mice Reveal That Differential Desensitization of ��2-Adrenergic Responses Is because of Ligand-selective Internalization

Although ligand-selective regulation of G protein-coupled receptor-mediated signaling and trafficking are well documented, little is known about whether ligand-selective effects occur on endogenous receptors or whether such effects modify the signaling response in physiologically relevant cells. Using a gene targeting approach, we generated a knock-in mouse line, in which N-terminal hemagglutinin epitope-tagged α_2A-adrenergic receptor (AR) expression was driven by the endogenous mouse α_2AAR gene locus. Exploiting this mouse line, we evaluated α_2AAR trafficking and α_2AAR-mediated inhibition of Ca²⁺ currents in native sympathetic neurons in response to clonidine and guanfacine, two drugs used for treatment of hypertension, attention deficit and hyperactivity disorder, and enhancement of analgesia through actions on the α_2AAR subtype. We discovered a more rapid desensitization of Ca²⁺ current suppression by clonidine than guanfacine, which paralleled a more marked receptor phosphorylation and endocytosis of α_2AAR evoked by clonidine than by guanfacine. Clonidine-induced α_2AAR desensitization, but not receptor phosphorylation, was attenuated by blockade of endocytosis with concanavalin A, indicating a critical role for internalization of α_2AAR in desensitization to this ligand. Our data on endogenous receptor-mediated signaling and trafficking in native cells reveal not only differential regulation of G protein-coupled receptor endocytosis by different ligands, but also a differential contribution of receptor endocytosis to signaling desensitization. Taken together, our data suggest that these HA-α_2AAR knock-in mice will serve as an important model in developing ligands to favor endocytosis or nonendocytosis of receptors, depending on the target cell and pathophysiology being addressed.G protein-coupled receptors (GPCRs)⁴ represent the largest family of cell surface receptors mediating responses to hormones, cytokines, neurotransmitters, and therapeutic agents (1). In addition to regulating downstream signaling, ligand binding to a receptor can initiate phosphorylation of the active conformation of the receptor by G protein receptor kinases (GRKs) and subsequent binding of arrestins, thus restricting the magnitude and duration of the ligand-evoked signaling responses (2, 3). Binding of arrestins to GPCRs also leads to GPCR internalization (4, 5), a process that has been proposed as a means to desensitize receptor signaling at the cell surface, resensitize receptors, and/or initiate intracellular signaling (6, 7).Different ligands are able to induce distinct signaling and internalization profiles of the same receptor (8-14). However, the lack of available tools to study trafficking of endogenous GPCRs in native target cells has limited our understanding of ligand-selective endocytosis profiles and the relative contribution of receptor endocytosis to desensitization in native biological settings.To specifically test hypotheses regarding ligand-selective effects on GPCR internalization, and functional consequences of this trafficking on signaling, we utilized a homologous recombination gene targeting strategy to introduce a hemagglutinin (HA) epitope-tagged wild type α_2A-adrenergic receptor (AR) into the mouse ADRA2A gene locus (“knock-in”). The α_2AAR is a prototypical GPCR that couples to the G_i/o subfamily of G proteins (15). Studies on genetically engineered mice made null or mutant for the α_2AAR have revealed that this subtype mediates the therapeutic effects of α₂-adrenergic agents on blood pressure, pain perception, volatile anesthetic sparing, analgesia, and working memory enhancement (16-18). Two classic α₂-ligands, clonidine and guanfacine, have been widely used to treat hypertension (19), attention deficit and hyperactivity disorder (20), and to elicit analgesia (19, 21) mediated via the α_2AAR. Clinically guanfacine has a much longer duration of action than clonidine (22-24); this longer duration of action cannot be accounted for by the pharmacokinetic profile of these agents in human beings, as both drugs have a half-life of 12-14 h (25, 26). Because ligand-induced desensitization and trafficking of GPCRs have been implicated as critical mechanisms for modulating response duration in vivo (3), one hypothesis underlying the difference in duration between clonidine and guanfacine is that clonidine provokes accelerated desensitization of the α_2AAR via one or several mechanisms, whereas guanfacine does not. Signaling desensitization in response to these two agonists has not been compared under the same experimental settings. To specifically test this hypothesis, we have exploited our HA-α_2AAR knock-in mice so that we could examine these properties of guanfacine and clonidine in native target cells.We compared internalization of the α_2AAR and inhibition of Ca²⁺ currents induced by clonidine and guanfacine in primary superior cervical ganglia (SCG) neurons, where the α_2AAR is the major adrenergic receptor subtype controlling norepinephrine release and sympathetic tone (17, 27). Our data revealed a differential regulation of α_2AAR trafficking and signaling duration by clonidine versus guanfacine, i.e. clonidine induced a more dramatic desensitization of the α_2AAR than guanfacine, and this desensitization was largely because of α_2AAR internalization. These studies reveal the powerful tool that the HA-α_2AAR knock-in mice provide for identifying endocytosis-dependent and -independent physiological phenomena for this receptor subtype as a first step in defining novel loci for therapeutic intervention in the α_2AAR signaling/trafficking cascade.     

Differential regulation of cell death programs in males and females by Poly (ADP-Ribose) Polymerase-1 and 17 β estradiol

Cell death can be divided into the anti-inflammatory process of apoptosis and the pro-inflammatory process of necrosis. Necrosis, as apoptosis, is a regulated form of cell death, and Poly-(ADP-Ribose) Polymerase-1 (PARP-1) and Receptor-Interacting Protein (RIP) 1/3 are major mediators. We previously showed that absence or inhibition of PARP-1 protects mice from nephritis, however only the male mice. We therefore hypothesized that there is an inherent difference in the cell death program between the sexes. We show here that in an immune-mediated nephritis model, female mice show increased apoptosis compared to male mice. Treatment of the male mice with estrogens induced apoptosis to levels similar to that in female mice and inhibited necrosis. Although PARP-1 was activated in both male and female mice, PARP-1 inhibition reduced necrosis only in the male mice. We also show that deletion of RIP-3 did not have a sex bias. We demonstrate here that male and female mice are prone to different types of cell death. Our data also suggest that estrogens and PARP-1 are two of the mediators of the sex-bias in cell death. We therefore propose that targeting cell death based on sex will lead to tailored and better treatments for each gender.     

Polo-like Kinase 2 (PLK2) Phosphorylates ��-Synuclein at Serine 129 in Central Nervous System

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of α-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to α-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited α-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in α-synuclein phosphorylation in central nervous system.The importance of α-synuclein to the pathogenesis of Parkinson disease (PD)⁴ and the related disorder, dementia with Lewy bodies (DLB), is suggested by its association with Lewy bodies and Lewy neurites, the inclusions that characterize these diseases (1–3), and demonstrated by the existence of mutations that cause syndromes mimicking sporadic PD and DLB (4–6). Furthermore, three separate mutations cause early onset forms of PD and DLB. It is particularly telling that duplications or triplications of the gene (7–9), which increase levels of α-synuclein with no alteration in sequence, also cause PD or DLB.α-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11, 12). Phosphorylation at tyrosine residues has been observed by some investigators (13, 14) but not by others (10–12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). In addition, p-Ser-129 was found to be the most extensive and consistent modification in a survey of synuclein in Lewy bodies (11). Results have been mixed from studies investigating the function of phosphorylation using S129A and S129D mutations to respectively block and mimic the modification. Although the phosphorylation mimic was associated with pathology in studies in Drosophila (16) and in transgenic mouse models (17, 18), studies using adeno-associated virus vectors to overexpress α-synuclein in rat substantia nigra found an exacerbation of pathology with the S129A mutation, whereas the S129D mutation was benign, if not protective (19). Interpretation of these studies is complicated by a recent study showing that the S129D and S129A mutations themselves have effects on the aggregation properties of α-synuclein independent of their effects on phosphorylation, with the S129A mutation stimulating fibril formation (20). Clearly, determination of the role of p-Ser-129 phosphorylation would be helped by identification of the responsible kinase. In addition, identification will provide a pathologically relevant way to increase phosphorylation in a cell or animal model.Several kinases have been proposed to phosphorylate α-synuclein, including casein kinases 1 and 2 (10, 12, 21) and members of the G-protein-coupled receptor kinase family (22). In this report, we offer evidence that a member of the polo-like kinase (PLK) family, PLK2 (or serum-inducible kinase, SNK), functions as an α-synuclein kinase. The ability of PLK2 to directly phosphorylate α-synuclein at Ser-129 is established by overexpression in cell culture and by in vitro reaction with the purified kinase. We show that PLK2 phosphorylates α-synuclein in cells, including primary neuronal cultures, using a series of kinase inhibitors as well as inhibition of expression with RNA interference. In addition, inhibitor and knock-out studies in mouse brain support a role for PLK2 as an α-synuclein kinase in vivo.     

Klebsiella Phage vB_KleM-RaK2 — A Giant Singleton Virus of the Family Myoviridae

At 346 kbp in size, the genome of a jumbo bacteriophage vB_KleM-RaK2 (RaK2) is the largest Klebsiella infecting myovirus genome sequenced to date. In total, 272 out of 534 RaK2 ORFs lack detectable database homologues. Based on the similarity to biologically defined proteins and/or MS/MS analysis, 117 of RaK2 ORFs were given a functional annotation, including 28 RaK2 ORFs coding for structural proteins that have no reliable homologues to annotated structural proteins in other organisms. The electron micrographs revealed elaborate spike-like structures on the tail fibers of Rak2, suggesting that this phage is an atypical myovirus. While head and tail proteins of RaK2 are mostly myoviridae-related, the bioinformatics analysis indicate that tail fibers/spikes of this phage are formed from podovirus-like peptides predominantly. Overall, these results provide evidence that bacteriophage RaK2 differs profoundly from previously studied viruses of the Myoviridae family.     

Pen2 and Presenilin-1 Modulate the Dynamic Equilibrium of Presenilin-1 and Presenilin-2 ��-Secretase Complexes

γ-Secretase is known to play a pivotal role in the pathogenesis of Alzheimer disease through production of amyloidogenic Aβ42 peptides. Early onset familial Alzheimer disease mutations in presenilin (PS), the catalytic core of γ-secretase, invariably increase the Aβ42:Aβ40 ratio. However, the mechanism by which these mutations affect γ-secretase complex formation and cleavage specificity is poorly understood. We show that our in vitro assay system recapitulates the effect of PS1 mutations on the Aβ42:Aβ40 ratio observed in cell and animal models. We have developed a series of small molecule affinity probes that allow us to characterize active γ-secretase complexes. Furthermore we reveal that the equilibrium of PS1- and PS2-containing active complexes is dynamic and altered by overexpression of Pen2 or PS1 mutants and that formation of PS2 complexes is positively correlated with increased Aβ42:Aβ40 ratios. These data suggest that perturbations to γ-secretase complex equilibrium can have a profound effect on enzyme activity and that increased PS2 complexes along with mutated PS1 complexes contribute to an increased Aβ42:Aβ40 ratio.β-Amyloid (Aβ)⁵ peptides are believed to play a causative role in Alzheimer disease (AD). Aβ peptides are generated from the processing of the amyloid precursor protein (APP) by two proteases, β-secretase and γ-secretase. Although γ-secretase generates heterogenous Aβ peptides ranging from 37 to 46 amino acids in length, significant work has focused mainly on the Aβ40 and Aβ42 peptides that are the major constituents of amyloid plaques. γ-Secretase is a multisubunit membrane aspartyl protease comprised of at least four known subunits: presenilin (PS), nicastrin (Nct), anterior pharynx-defective (Aph), and presenilin enhancer 2 (Pen2). Presenilin is thought to contain the catalytic core of the complex (1–4), whereas Aph and Nct play critical roles in the assembly, trafficking, and stability of γ-secretase as well as substrate recognition (5, 6). Lastly Pen2 facilitates the endoproteolysis of PS into its N-terminal (NTF) and C-terminal (CTF) fragments thereby yielding a catalytically competent enzyme (5, 7–10). All four proteins (PS, Nct, Aph1, and Pen2) are obligatory for γ-secretase activity in cell and animal models (11, 12). There are two homologs of PS, PS1 and PS2, and three isoforms of Aph1, Aph1aS, Aph1aL, and Aph1b. At least six active γ-secretase complexes have been reported (two presenilins × three Aph1s) (13, 14). The sum of apparent molecular masses of the four proteins (PS1-NTF/CTF ≈ 53 kDa, Nct ≈ 120 kDa, Aph1 ≈ 30 kDa, and Pen2 ≈ 10kDa) is ∼200 kDa. However, active γ-secretase complexes of varying sizes, ranging from 250 to 2000 kDa, have been reported (15–19). Recently a study suggested that the γ-secretase complex contains only one of each subunit (20). Collectively these studies suggest that a four-protein complex around 200–250 kDa may be the minimal functional γ-secretase unit with additional cofactors and/or varying stoichiometry of subunits existing in the high molecular weight γ-secretase complexes. CD147 and TMP21 have been found to be associated with the γ-secretase complex (21, 22); however, their role in the regulation of γ-secretase has been controversial (23, 24).Mutations of PS1 or PS2 are associated with familial early onset AD (FAD), although it is debatable whether these familial PS mutations act as “gain or loss of function” alterations in regard to γ-secretase activity (25–27). Regardless the overall outcome of these mutations is an increased ratio of Aβ42:Aβ40. Clearly these mutations differentially affect γ-secretase activity for the production of Aβ40 and Aβ42. Despite intensive studies of Aβ peptides and γ-secretase, the molecular mechanism controlling the specificity of γ-secretase activity for Aβ40 and Aβ42 production has not been resolved. It has been found that PS1 mutations affect the formation of γ-secretase complexes (28). However, the precise mechanism by which individual subunits alter the dynamics of γ-secretase complex formation and activity is largely unresolved. A better mechanistic understanding of γ-secretase activity associated with FAD mutations has been hindered by the lack of suitable assays and probes that are necessary to recapitulate the effect of these mutations seen in cell models and to characterize the active γ-secretase complex.In our present studies, we have determined the overall effect of Pen2 and PS1 expression on the dynamics of PS1- and PS2-containing complexes and their association with γ-secretase activity. Using newly developed biotinylated small molecular probes and activity assays, we revealed that expression of Pen2 or PS1 FAD mutants markedly shifts the equilibrium of PS1-containing active complexes to that of PS2-containing complexes and results in an overall increase in the Aβ42:Aβ40 ratio in both stable cell lines and animal models. Our studies indicate that perturbations to the equilibrium of active γ-secretase complexes by an individual subunit can greatly affect the activity of the enzyme. Moreover they serve as further evidence that there are multiple and distinct γ-secretase complexes that can exist within the same cells and that their equilibrium is dynamic. Additionally the affinity probes developed here will facilitate further study of the expression and composition of endogenous active γ-secretase from a variety of model systems.     

In Situ OH Generation from O2 ? and H2O2 Plays a Critical Role in Plasma-Induced Cell Death

Reactive oxygen and nitrogen species produced by cold atmospheric plasma (CAP) are considered to be the most important species for biomedical applications, including cancer treatment. However, it is not known which species exert the greatest biological effects, and the nature of their interactions with tumor cells remains ill-defined. These questions were addressed in the present study by exposing human mesenchymal stromal and LP-1 cells to reactive oxygen and nitrogen species produced by CAP and evaluating cell viability. Superoxide anion (O₂ ⁻) and hydrogen peroxide (H₂O₂) were the two major species present in plasma, but their respective concentrations were not sufficient to cause cell death when used in isolation; however, in the presence of iron, both species enhanced the cell death-inducing effects of plasma. We propose that iron containing proteins in cells catalyze O₂ ⁻ and H₂O₂ into the highly reactive OH radical that can induce cell death. The results demonstrate how reactive species are transferred to liquid and converted into the OH radical to mediate cytotoxicity and provide mechanistic insight into the molecular mechanisms underlying tumor cell death by plasma treatment.     

T Cell Receptor-dependent Tyrosine Phosphorylation of ��2-Chimaerin Modulates Its Rac-GAP Function in T Cells

The actin cytoskeleton has an important role in the organization and function of the immune synapse during antigen recognition. Dynamic rearrangement of the actin cytoskeleton in response to T cell receptor (TCR) triggering requires the coordinated activation of Rho family GTPases that cycle between active and inactive conformations. This is controlled by GTPase-activating proteins (GAP), which regulate inactivation of Rho GTPases, and guanine exchange factors, which mediate their activation. Whereas much attention has centered on guanine exchange factors for Rho GTPases in T cell activation, the identity and functional roles of the GAP in this process are largely unknown. We previously reported β2-chimaerin as a diacylglycerol-regulated Rac-GAP that is expressed in T cells. We now demonstrate Lck-dependent phosphorylation of β2-chimaerin in response to TCR triggering. We identify Tyr-153 as the Lck-dependent phosphorylation residue and show that its phosphorylation negatively regulates membrane stabilization of β2-chimaerin, decreasing its GAP activity to Rac. This study establishes the existence of TCR-dependent regulation of β2-chimaerin and identifies a novel mechanism for its inactivation.T cell activation requires presentation of an antigen by antigen-presenting cells (APC)² to the T cell receptor (TCR); this event involves the reorganization of several scaffolds and signaling proteins, leading to formation of the immunological synapse (IS) (1). Correct protein redistribution during synapse formation is critical for an efficient T cell response, and it is largely regulated by actin polymerization at the T cell/APC contact site as a result of TCR-regulated Rac-dependent signals (2, 3). Like other Rho GTPases, Rac cycles between a GTP-bound active state and a GDP-bound inactive state. This continuous recycling is regulated by the concerted action of two proteins as follows: GEF, which activates Rac by mediating GDP/GTP exchange (4), and GAP, which induces Rac inactivation by accelerating intrinsic Rac GTPase activity, converting GTP to GDP (5).Vav-1 is the best studied GEF for Rac, and it has critical roles in T cell-dependent functions (6). In naive, unstimulated T cells, Vav-1 is in an inactive state through autoinhibition, as the GEF domain is blocked by the N-terminal region (7). This autoinhibition is relieved by TCR-mediated tyrosine phosphorylation (8, 9). Thymocytes from Vav-1-deficient mice have a developmental block, and their mature T cells show severe defects in IS formation, as well as reduced Ca²⁺ influx, IL-2 production, T cell proliferation, and cytotoxic activity (10–13). Although several studies have shown a key role for Vav-1, the mechanisms that govern Rac inactivation downstream of the TCR remain elusive.The chimaerins are a family of Rho-GAP, with specific activity for Rac. In addition to their catalytic domain, they have an N-terminal SH2 domain and a C1 domain required for interaction with the lipid messenger diacylglycerol (DAG) and with phorbol esters (14). There are two mammalian chimaerin genes (CHN1 and CHN2), which encode the full-lengthα2-(ARHGAP2) and β2-chimaerins (ARHGAP3), and at least one splice variant each (α1 and β1) that lack the SH2 domain. The α-chimaerins are expressed abundantly in brain and are linked to neuritogenesis and axon guidance (15–20). β2-Chimaerin expression is ubiquitous (21) and is involved in EGF-dependent Rac regulation (22, 23). Experiments in T cells showed that β2-chimaerin participates in chemokine-dependent regulation of T cell migration and adhesion (24). A very recent study implicates chimaerins in the modulation of Rac activity during T cell synapse formation, suggesting that this protein family contributes to DAG-mediated regulation of cytoskeletal remodeling during T cell activation (25).Determination of the β2-chimaerin crystal structure provided important clues regarding its mechanism of action. In the absence of stimulation, the protein is in an inactive state in which the N-terminal domain maintains a “closed” conformation, blocking Rac binding and concealing the C1 domain (26). These structural data were fully supported by experiments in live T lymphocytes showing that phorbol myristate acetate (PMA)-dependent translocation of β2-chimaerin was less effective than that of its isolated C1 domain (24). These data not only confirmed the lack of accessibility of the β2-chimaerin C1 domain but also suggested that there are negative regulatory mechanisms that promote β2-chimaerin release from the membrane.DAG-dependent signaling is critical for the modulation of T cell functions, by virtue of its ability to bind and regulate C1 domain-containing proteins such as protein kinase Cθ, protein kinase D, and RasGRP1 (27). An important issue is to determine how the different DAG-binding proteins discriminate between distinct DAG pools, and how DAG activates certain C1-containing proteins and not others. Some mechanisms that allow discrimination between DAG receptors include the distinct affinity of C1 domains for different DAG pools, association of C1 domain-containing proteins to specific scaffolds, and/or structural determinants in these proteins that limit C1 domain accessibility to membrane DAG (28–30).To explore the events that contribute to the specific regulation of β2-chimaerin, we studied β2-chimaerin phosphorylation in the context of TCR stimulation. We show that β2-chimaerin is phosphorylated in tyrosine residues after TCR stimulation, and we identify Lck as the Tyr kinase responsible for this phosphorylation. Generation of point mutants identified Tyr-153, at the hinge of the SH2 and C1 domains, as the main tyrosine residue phosphorylated in response to TCR stimulation. Cells expressing a β2-chimaerin mutant defective for Tyr-153 phosphorylation show anomalies in TCR clustering, conjugate formation, NF-AT activation, and IL-2 production that correlate with elevated Rac-GAP activity in this mutant. Subcellular localization analysis of the β2-chimaerin mutants reveals that impairment of β2-chimaerin phosphorylation at Tyr-153 promotes C1-mediated β2-chimaerin stabilization at the plasma membrane, providing a mechanistic explanation for its higher Rac-GAP activity. In summary, our results demonstrate for the first time that tyrosine kinase-mediated negative regulation of β2-chimaerin is elicited by physiological stimulation in T lymphocytes, and suggest that TCR stimulation provides both positive and negative signals for β2-chimaerin activation.     

Does Leaf Position within a Canopy Affect Acclimation of Photosynthesis to Elevated CO2? : Analysis of a Wheat Crop under Free-Air CO2 Enrichment

Previous studies of photosynthetic acclimation to elevated CO₂ have focused on the most recently expanded, sunlit leaves in the canopy. We examined acclimation in a vertical profile of leaves through a canopy of wheat (Triticum aestivum L.). The crop was grown at an elevated CO₂ partial pressure of 55 Pa within a replicated field experiment using free-air CO₂ enrichment. Gas exchange was used to estimate in vivo carboxylation capacity and the maximum rate of ribulose-1,5-bisphosphate-limited photosynthesis. Net photosynthetic CO₂ uptake was measured for leaves in situ within the canopy. Leaf contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), light-harvesting-complex (LHC) proteins, and total N were determined. Elevated CO₂ did not affect carboxylation capacity in the most recently expanded leaves but led to a decrease in lower, shaded leaves during grain development. Despite this acclimation, in situ photosynthetic CO₂ uptake remained higher under elevated CO₂. Acclimation at elevated CO₂ was accompanied by decreases in both Rubisco and total leaf N contents and an increase in LHC content. Elevated CO₂ led to a larger increase in LHC/Rubisco in lower canopy leaves than in the uppermost leaf. Acclimation of leaf photosynthesis to elevated CO₂ therefore depended on both vertical position within the canopy and the developmental stage.     

Aquaporin-2 in the ��-omics�� Era

Vasopressin controls renal water excretion largely through actions to regulate the water channel aquaporin-2 in collecting duct principal cells. Our knowledge of the mechanisms involved has increased markedly in recent years with the advent of methods for large-scale systems-level profiling such as protein mass spectrometry, yeast two-hybrid analysis, and oligonucleotide microarrays. Here we review this progress.Regulation of water excretion by the kidney is one of the most visible aspects of everyday physiology. An outdoor tennis game on a hot summer day can result in substantial water losses by sweating, and the kidneys respond by reducing water excretion. In contrast, excessive intake of water, a frequent occurrence in everyday life, results in excretion of copious amounts of clear urine. These responses serve to exact tight control on the tonicity of body fluids, maintaining serum osmolality in the range of 290–294 mosmol/kg of H₂O through the regulated return of water from the pro-urine in the renal collecting ducts to the bloodstream.The importance of this process is highlighted when the regulation fails. For example, polyuria (rapid uncontrolled excretion of water) is a sometimes devastating consequence of lithium therapy for bipolar disorder. On the other side of the coin are water balance disorders that result from excessive renal water retention causing systemic hypo-osmolality or hyponatremia. Hyponatremia due to excessive water retention can be seen with severe congestive heart failure, hepatic cirrhosis, and the syndrome of inappropriate antidiuresis.The chief regulator of water excretion is the peptide hormone AVP,² whereas the chief molecular target for regulation is the water channel AQP2. In this minireview, we describe new progress in the understanding of the molecular mechanisms involved in regulation of AQP2 by AVP in collecting duct cells, with emphasis on new information derived from “systems-level” approaches involving large-scale profiling and screening techniques such as oligonucleotide arrays, protein mass spectrometry, and yeast two-hybrid analysis. Most of the progress with these techniques is in the identification of individual molecules involved in AVP signaling and binding interactions with AQP2. Additional related issues are addressed in several recent reviews (1–4).     

Unlipidated Outer Membrane Protein Omp16 (U-Omp16) from Brucella spp. as Nasal Adjuvant Induces a Th1 Immune Response and Modulates the Th2 Allergic Response to Cow’s Milk Proteins

The discovery of novel mucosal adjuvants will help to develop new formulations to control infectious and allergic diseases. In this work we demonstrate that U-Omp16 from Brucella spp. delivered by the nasal route (i.n.) induced an inflammatory immune response in bronchoalveolar lavage (BAL) and lung tissues. Nasal co-administration of U-Omp16 with the model antigen (Ag) ovalbumin (OVA) increased the amount of Ag in lung tissues and induced OVA-specific systemic IgG and T helper (Th) 1 immune responses. The usefulness of U-Omp16 was also assessed in a mouse model of food allergy. U-Omp16 i.n. administration during sensitization ameliorated the hypersensitivity responses of sensitized mice upon oral exposure to Cow’s Milk Protein (CMP), decreased clinical signs, reduced anti-CMP IgE serum antibodies and modulated the Th2 response in favor of Th1 immunity. Thus, U-Omp16 could be used as a broad Th1 mucosal adjuvant for different Ag formulations.