Cpt-Cgmp Is A New Ligand Of Epithelial Sodium Channels

Epithelial sodium channels (ENaC) are localized at the apical membrane of the epithelium, and are responsible for salt and fluid reabsorption. Renal ENaC takes up salt, thereby controlling salt content in serum. Loss-of-function ENaC mutations lead to low blood pressure due to salt-wasting, while gain-of-function mutations cause impaired sodium excretion and subsequent hypertension as well as hypokalemia. ENaC activity is regulated by intracellular and extracellular signals, including hormones, neurotransmitters, protein kinases, and small compounds. Cyclic nucleotides are broadly involved in stimulating protein kinase A and protein kinase G signaling pathways, and, surprisingly, also appear to have a role in regulating ENaC. Increasing evidence suggests that the cGMP analog, CPT-cGMP, activates αβγ-ENaC activity reversibly through an extracellular pathway in a dose-dependent manner. Furthermore, the parachlorophenylthio moiety and ribose 2’-hydroxy group of CPT-cGMP are essential for facilitating the opening of ENaC channels by this compound. Serving as an extracellular ligand, CPT-cGMP eliminates sodium self-inhibition, which is a novel mechanism for stimulating salt reabsorption in parallel to the traditional NO/cGMP/PKG signal pathway. In conclusion, ENaC may be a druggable target for CPT-cGMP, leading to treatments for kidney malfunctions in salt reabsorption

Proteolytic Regulation Of Epithelial Sodium Channels By Urokinase Plasminogen Activator: Cutting Edge And Cleavage Sites

Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at (177GR↓KR180) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically \ silent\ ) channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions

Isolation and complete genomic characterization of H1N1 subtype swine influenza viruses in southern China through the 2009 pandemic

<p>Abstract</p> <p>Background</p> <p>The swine influenza (SI) is an infectious disease of swine and human. The novel swine-origin influenza A (H1N1) that emerged from April 2009 in Mexico spread rapidly and caused a human pandemic globally. To determine whether the tremendous virus had existed in or transmitted to pigs in southern China, eight H1N1 influenza strains were identified from pigs of Guangdong province during 2008-2009.</p> <p>Results</p> <p>Based on the homology and phylogenetic analyses of the nucleotide sequences of each gene segments, the isolates were confirmed to belong to the classical SI group, with HA, NP and NS most similar to 2009 human-like H1N1 influenza virus lineages. All of the eight strains were low pathogenic influenza viruses, had the same host range, and not sensitive to class of antiviral drugs.</p> <p>Conclusions</p> <p>This study provides the evidence that there is no 2009 H1N1-like virus emerged in southern China, but the importance of swine influenza virus surveillance in China should be given a high priority.</p

Structural and Functional Characterization of Two Alternative Splicing Variants of Mouse Endothelial Cell-Specific Chemotaxis Regulator (ECSCR)

Endothelial cells (ECs) that line the lumen of blood vessels are important players in blood vessel formation, and EC migration is a key component of the angiogenic process. Thus, identification of genes that are specifically or preferentially expressed in vascular ECs and in-depth understanding of their biological functions may lead to discovery of new therapeutic targets. We have previously reported molecular characterization of human endothelial cell-specific molecule 2 (ECSM2)/endothelial cell-specific chemotaxis regulator (ECSCR). In the present study, we cloned two mouse full-length cDNAs by RT-PCR, which encode two putative ECSCR isoform precursors with considerable homology to the human ECSCR. Nucleotide sequence and exon-intron junction analyses suggested that they are alternative splicing variants (ECSCR isoform-1 and -2), differing from each other in the first and second exons. Quantitative RT-PCR results revealed that isoform-2 is the predominant form, which was most abundant in heart, lung, and muscles, and moderately abundant in uterus and testis. In contrast, the expression of isoform-1 seemed to be more enriched in testis. To further explore their potential cellular functions, we expressed GFP- and FLAG-tagged ECSCR isoforms, respectively, in an ECSCR deficient cell line (HEK293). Interestingly, the actual sizes of either ECSCR-GFP or -FLAG fusion proteins detected by immunoblotting are much larger than their predicted sizes, suggesting that both isoforms are glycoproteins. Fluorescence microscopy revealed that both ECSCR isoforms are localized at the cell surface, which is consistent with the structural prediction. Finally, we performed cell migration assays using mouse endothelial MS1 cells overexpressing GFP alone, isoform-1-GFP, and isoform-2-GFP, respectively. Our results showed that both isoforms significantly inhibited vascular epidermal growth factor (VEGF)-induced cell migration. Taken together, we have provided several lines of experimental evidence that two mouse ECSCR splicing variants/isoform precursors exist. They are differentially expressed in a variety of tissue types and likely involved in modulation of vascular EC migration. We have also defined the gene structure of mouse ECSCR using bioinformatics tools, which provides new information towards a better understanding of alternative splicing of ECSCR

Modeling Rett Syndrome Using TALEN-Edited MECP2 Mutant Cynomolgus Monkeys

Gene-editing technologies have made it feasible to create nonhuman primate models for human genetic disorders. Here, we report detailed genotypes and phenotypes of TALEN-edited MECP2 mutant cynomolgus monkeys serving as a model for a neurodevelopmental disorder, Rett syndrome (RTT), which is caused by loss-of-function mutations in the human MECP2 gene. Male mutant monkeys were embryonic lethal, reiterating that RTT is a disease of females. Through a battery of behavioral analyses, including primate-unique eye-tracking tests, in combination with brain imaging via MRI, we found a series of physiological, behavioral, and structural abnormalities resembling clinical manifestations of RTT. Moreover, blood transcriptome profiling revealed that mutant monkeys resembled RTT patients in immune gene dysregulation. Taken together, the stark similarity in phenotype and/or endophenotype between monkeys and patients suggested that gene-edited RTT founder monkeys would be of value for disease mechanistic studies as well as development of potential therapeutic interventions for RTT

A Computational Framework For The 3D Structure Determination Of Viruses With Unknown Symmetry

The protein shell of all virus structures resolved to this date exhibit some form of symmetry, most of them are spherical viruses with icosahedral symmetry; the study of viruses whose symmetry is not known, of structures which do not exhibit any symmetry, as well as the study of the genetic material of a virus are considerably more challenging. Increasing the resolution of the structure determination and solving structures with no symmetry represents a quantum leap in virus structure determination based upon electron microscopy. Computing is a major component of the structure determination process. Nowadays it is not feasible to increase the resolution of the structure determination of large macromolecules like viruses or to solve structures with no symmetry without novel parallel algorithms and environments enabling structural biologists to use parallel systems, clusters of workstations, or providing access to grid computing. © 2003 Elsevier Inc. All rights reserved

Rotational And Translational Alignment Errors In 3D Reconstruction Of Virus Structures At High Resolution

The 3D reconstruction of virus structures at high resolution using CryoTEM data requires a very accurate rotational and translational alignment of individual views obtained experimentally. We discuss the geometrical foundations and the computational problems raised by rotational and translational alignment. We also outline the basic ideas for CTF correction. Copyright © 2006 Inderscience Enterprises Ltd

Rotational And Translational Alignment Errors In 3D Reconstruction Of Virus Structures At High Resolution

3D reconstruction of virus structures at high resolution using CryoTEM data requires a very accurate rotational and translational alignment of individual views obtained experimentally. We discuss the geometrical foundations and the computational problems raised by rotational and translational alignment. We also outline the basic ideas for CTF correction. © 2005 IEEE