Publications by authors named "Ruifeng Zhou"

23 Publications

  • Page 1 of 1

Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model.

bioRxiv 2021 Apr 4. Epub 2021 Apr 4.

In the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males compared with females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8-10 weeks of age) were inoculated intranasally with 10 TCID of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developing more extensive pneumonia as noted on chest computed tomography, and recovering more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including IFNb and TNFa, were comparable between the sexes. However, during the recovery phase of infection, females mounted two-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole inactivated SARS-CoV-2 and mutant S-RBDs, as well as virus neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2 associated sex differences seen in the human population.
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http://dx.doi.org/10.1101/2021.04.02.438292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020969PMC
April 2021

Potential energy shift of the Fermi level at plasmonic structures for light-energy conversion determined by graphene-based Raman measurements.

J Chem Phys 2020 Mar;152(12):124702

Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.

Single layer graphene was used to determine the electrochemical potential of plasmonic nano-structures for photoelectrochemical energy conversions. From electrochemical Raman measurements of the graphene layer under near-infrared light, illumination has revealed the relationship between the photoenergy conversion ability and the Fermi level of the plasmonic structure. The determination is based on in situ monitoring of G and 2D Raman bands of the graphene layer on plasmonic structures. The correlation plots of G and 2D bands show the dependence on the photoconversion ability. The present electrochemical Raman measurements provide detailed understanding of the plasmon-induced charge transfer process for further developments on the ability.
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http://dx.doi.org/10.1063/1.5143560DOI Listing
March 2020

Efficacy and Tolerability of Lopinavir/Ritonavir- and Efavirenz-Based Initial Antiretroviral Therapy in HIV-1-Infected Patients in a Tertiary Care Hospital in Beijing, China.

Front Pharmacol 2019 12;10:1472. Epub 2019 Dec 12.

Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing, China.

Lopinavir/ritonavir (LPV/r) is a major antiretroviral treatment in China, but little is known about the performance of first-line LPV/r-based regimen in treatment-naïve patients with human immunodeficiency virus type 1 (HIV-1) infection. This study aims to assess the efficacy and adverse effect events of LPV/r plus lamivudine and tenofovir or zidovudine as an initial antiretroviral treatment in HIV-1-infected individuals for whom cannot take efavirenz (EFV) or is allergic to EFV. We performed a retrospective study of patients registering with the China's National Free Antiretroviral Treatment Program from July 2012 to January 2017, followed at a tertiary care hospital in Beijing, China. The primary outcome was the proportion of subjects with HIV-1 RNA ≤40 copies/ml at 6 and 24 months of treatment. We assessed the immunological response and adverse events. In total, 4,862 patients were enrolled in the study and 237 were eligible for analysis in each study arm. During the first six months, virological suppression was better with the LPV/r-based regimen than with the EFV-based regimen (93.80 vs 87.80% for < 0.05). Viral suppression rates continued to increase until 12 months, remain steady thereafter until 24 months, for both groups. The multilevel analysis revealed that patients in the LPV/r group were more likely to display improvements in CD4 T-cell count over time than those in the EFV group ( < 0.001). Grade 3 or 4 laboratory adverse events were observed in 14 patients (5.91%) from the LPV/r group and three patients (1.20%) in EFV group. Our findings demonstrate that LPV/r-containing regimens are effective and well-tolerated in Chinese treatment-naïve patients with HIV-1 infection.
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http://dx.doi.org/10.3389/fphar.2019.01472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920196PMC
December 2019

Nonzero Wavevector Excitation of Graphene by Localized Surface Plasmons.

Nano Lett 2019 11 2;19(11):7887-7894. Epub 2019 Oct 2.

Department of Chemistry, Faculty of Science , Hokkaido University , Sapporo 060-0810 , Japan.

Electrochemical surface-enhanced Raman scattering measurements of single layer graphene provide unique information on resonant excitation induced by localized surface plasmons under controlled electron or hole doping. The highly confined electromagnetic field from the LSPs of the Au nanodimer structures prepared on defect-free graphene can generate holes and electrons of the electrochemical potentials beyond the limit of far-field light illumination. The electrochemical in situ SERS spectra prove nonzero wavevector excitation through the observation of normally forbidden Raman bands in graphene. The present findings point to a novel approach to breaking the limit of optoelectronic interactions and photochemical reactions of graphene and other semiconductors.
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http://dx.doi.org/10.1021/acs.nanolett.9b02947DOI Listing
November 2019

Revealing High Oxygen Evolution Catalytic Activity of Fluorine-Doped Carbon in Alkaline Media.

Materials (Basel) 2019 Jan 10;12(2). Epub 2019 Jan 10.

Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan.

Oxygen evolution reactions (OER) are important reactions for energy conversion. Metal-free carbon-based catalysts potentially contribute to the catalytic materials for OER. However, it has been difficult to understand the intrinsic catalytic activity of carbon materials, due to catalyst decomposition over the course of long-term reactions. Here, we report high oxygen evolution reaction catalytic activity of F-doped carbon in alkaline media. Intrinsic OER activity was evaluated from a combination of measurements using a rotating disk electrode and O₂ sensor. The F-doped carbon catalyst is a highly active catalyst, comparable to state-of-the-art precious-metal-based catalysts such as RuO₂.
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http://dx.doi.org/10.3390/ma12020211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356676PMC
January 2019

Sensitive Raman Probe of Electronic Interactions between Monolayer Graphene and Substrate under Electrochemical Potential Control.

ACS Omega 2018 Feb 27;3(2):2322-2328. Epub 2018 Feb 27.

Department of Chemistry, Faculty of Science, Hokkaido University, N10W8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.

In situ electrochemical Raman spectroscopic measurements of defect-free monolayer graphene on various substrates were performed under electrochemical potential control. The G and 2D Raman band wavenumbers (ω, ω) of graphene were found to depend upon the electrochemical potential, i.e., the charge density of graphene. The values of ω and ω also varied depending on the choice of substrates. On metal substrates where graphene was synthesized by chemical vapor deposition, a strong blue shift of ω was induced, which could not account for the strain and charge doping. We attributed the blue shift of ω to a change in the electronic properties of graphene induced by distinct electronic interactions with the metal substrates. To explain the unique characteristics in the Raman spectrum of graphene on various substrates, a novel mechanism is proposed considering reduction of the Fermi velocity in graphene owing to dielectric screening from the metal substrates.
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http://dx.doi.org/10.1021/acsomega.7b01928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6641367PMC
February 2018

An Fe/N co-doped graphitic carbon bulb for high-performance oxygen reduction reaction.

Chem Commun (Camb) 2015 May;51(35):7516-9

School of Chemical Engineering, University of Adelaide, Adelaide SA 5005, Australia.

An Fe/N co-doped graphitic carbon bulb is synthesized by Prussian blue with a pyrolysis temperature as low as 550 °C. Fe facilitates the formation of a graphitic structure, while low temperature guarantees high level of nitrogen. The product shows excellent oxygen reduction reaction catalytic activity in both alkaline and acid electrolytes.
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http://dx.doi.org/10.1039/c5cc00995bDOI Listing
May 2015

Hierarchical mesoporous yolk-shell structured carbonaceous nanospheres for high performance electrochemical capacitive energy storage.

Chem Commun (Camb) 2015 Feb;51(13):2518-21

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia.

Hierarchical mesoporous yolk-shell structured carbon nanospheres (YSCNs) with an ordered mesoporous carbon core and a microporous carbon shell show excellent electrochemical performance with a maximal specific capacitance of 159 F g(-1).
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http://dx.doi.org/10.1039/c4cc09366fDOI Listing
February 2015

Ubiquitin-specific peptidase 8 (USP8) regulates endosomal trafficking of the epithelial Na+ channel.

J Biol Chem 2013 Feb 7;288(8):5389-97. Epub 2013 Jan 7.

Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA.

Ubiquitination plays a key role in trafficking of the epithelial Na(+) channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na(+) absorption.
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http://dx.doi.org/10.1074/jbc.M112.425272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581384PMC
February 2013

Regulation of epithelial sodium channel trafficking by proprotein convertase subtilisin/kexin type 9 (PCSK9).

J Biol Chem 2012 Jun 9;287(23):19266-74. Epub 2012 Apr 9.

Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA.

The epithelial Na(+) channel (ENaC) is critical for Na(+) homeostasis and blood pressure control. Defects in its regulation cause inherited forms of hypertension and hypotension. Previous work found that ENaC gating is regulated by proteases through cleavage of the extracellular domains of the α and γ subunits. Here we tested the hypothesis that ENaC is regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9), a protease that modulates the risk of cardiovascular disease. PCSK9 reduced ENaC current in Xenopus oocytes and in epithelia. This occurred through a decrease in ENaC protein at the cell surface and in the total cellular pool, an effect that did not require the catalytic activity of PCSK9. PCSK9 interacted with all three ENaC subunits and decreased their trafficking to the cell surface by increasing proteasomal degradation. In contrast to its previously reported effects on the LDL receptor, PCSK9 did not alter ENaC endocytosis or degradation of the pool of ENaC at the cell surface. These results support a role for PCSK9 in the regulation of ENaC trafficking in the biosynthetic pathway, likely by increasing endoplasmic reticulum-associated degradation. By reducing ENaC channel number, PCSK9 could modulate epithelial Na(+) absorption, a major contributor to blood pressure control.
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http://dx.doi.org/10.1074/jbc.M112.363382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365958PMC
June 2012

A graphene oxide-carbon nanotube grid for high-resolution transmission electron microscopy of nanomaterials.

Nanotechnology 2011 Sep 31;22(38):385704. Epub 2011 Aug 31.

Department of Physics, Tsinghua-Foxconn Nanotechnology Research Centre, Tsinghua University, Beijing, People's Republic of China.

A novel grid for use in transmission electron microscopy is developed. The supporting film of the grid is composed of thin graphene oxide films overlying a super-aligned carbon nanotube network. The composite film combines the advantages of graphene oxide and carbon nanotube networks and has the following properties: it is ultra-thin, it has a large flat and smooth effective supporting area with a homogeneous amorphous appearance, high stability, and good conductivity. The graphene oxide-carbon nanotube grid has a distinct advantage when characterizing the fine structure of a mass of nanomaterials over conventional amorphous carbon grids. Clear high-resolution transmission electron microscopy images of various nanomaterials are obtained easily using the new grids.
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http://dx.doi.org/10.1088/0957-4484/22/38/385704DOI Listing
September 2011

Scratch-resistant, highly conductive, and high-strength carbon nanotube-based composite yarns.

ACS Nano 2010 Oct;4(10):5827-34

Department of Physics and Tsinghua-Foxconn Nanotechnology Research Center, Tsinghua University, Beijing 100084, China.

High-strength and conductive carbon nanotube (CNT) yarns are very attractive in many potential applications. However, there is a difficulty when simultaneously enhancing the strength and conductivity of CNT yarns. Adding some polymers into CNT yarns to enhance their strength will decrease their conductivity, while treating them in acid or coating them with metal nanoparticles to enhance their conductivity will reduce their strength. To overcome this difficulty, here we report a method to make high-strength and highly conductive CNT-based composite yarns by using a continuous superaligned CNT (SACNT) yarn as a conductive framework and then inserting polyvinyl alcohol (PVA) into the intertube spaces of the framework through PVA/dimethyl sulphoxide solution to enhance the strength of yarns. The as-produced CNT/PVA composite yarns possess very high tensile strengths up to 2.0 GPa and Young's moduli more than 120 GPa, much higher than those of the CNT/PVA yarns reported. The electric conductivity of as-produced composite yarns is as high as 9.2 × 10(4) S/m, comparable to HNO(3)-treated or Au nanoparticle-coated CNT yarns. These composite yarns are flexible, lightweight, scratch-resistant, very stable in the lab environment, and resistant to extremely humid ambient and as a result can be woven into high-strength and heatable fabrics, showing potential applications in flexible heaters, bullet-proof vests, radiation protection suits, and spacesuits.
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http://dx.doi.org/10.1021/nn1017318DOI Listing
October 2010

High-performance supercapacitors using a nanoporous current collector made from super-aligned carbon nanotubes.

Nanotechnology 2010 Aug 4;21(34):345701. Epub 2010 Aug 4.

Department of Physics, Tsinghua University, Beijing, People's Republic of China.

Nanoporous current collectors for supercapacitors have been fabricated by cross-stacking super-aligned carbon nanotube (SACNT) films as a replacement for heavy conventional metallic current collectors. The CNT-film current collectors have good conductivity, extremely low density (27 microg cm(-2)), high specific surface area, excellent flexibility and good electrochemical stability. Nanosized active materials such as NiO, Co(3)O(4) or Mn(2)O(3) nanoparticles can be directly synthesized on the SACNT films by a straightforward one-step, in situ decomposition strategy that is both efficient and environmentally friendly. These composite films can be integrated into a pseudo-capacitor that does not use metallic current collectors, but nevertheless shows very good performance, including high specific capacitance (approximately 500 F g(-1), including the current collector mass), reliable electrochemical stability (<4.5% degradation in 2500 cycles) and a very high rate capability (245 F g(-1) at 155 A g(-1)).
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http://dx.doi.org/10.1088/0957-4484/21/34/345701DOI Listing
August 2010

Hrs controls sorting of the epithelial Na+ channel between endosomal degradation and recycling pathways.

J Biol Chem 2010 Oct 30;285(40):30523-30. Epub 2010 Jul 30.

Department of Internal Medicine, Roy J and Lucille A Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.

Epithelial Na(+) absorption is regulated by Nedd4-2, an E3 ubiquitin ligase that reduces expression of the epithelial Na(+) channel (ENaC) at the cell surface. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 functions through two distinct effects on trafficking, enhancing both ENaC endocytosis and ENaC degradation in lysosomes. To investigate the mechanism by which Nedd4-2 targets ENaC to lysosomes, we tested the role of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of the endosomal sorting complexes required for transport (ESCRT)-0 complex. We found that α-, β-, and γENaC each interact with Hrs. These interactions were enhanced by Nedd4-2 and were dependent on the catalytic function of Nedd4-2 as well as its WW domains. Mutation of ENaC PY motifs, responsible for inherited hypertension (Liddle syndrome), decreased Hrs binding to ENaC. Moreover, binding of ENaC to Hrs was reduced by dexamethasone/serum- and glucocorticoid-inducible kinase and cAMP, which are signaling pathways that inhibit Nedd4-2. Nedd4-2 bound to Hrs and catalyzed Hrs ubiquitination but did not alter Hrs protein levels. Expression of a dominant negative Hrs lacking its ubiquitin-interacting motif (Hrs-ΔUIM) increased ENaC surface expression and current. This occurred through reduced degradation of the cell surface pool of proteolytically activated ENaC, which enhanced its recycling to the cell surface. In contrast, Hrs-ΔUIM had no effect on degradation of uncleaved inactive channels. The data support a model in which Nedd4-2 induces binding of ENaC to Hrs, which mediates the sorting decision between ENaC degradation and recycling.
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http://dx.doi.org/10.1074/jbc.M110.150755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2945546PMC
October 2010

Carbon nanotube yarns with high tensile strength made by a twisting and shrinking method.

Nanotechnology 2010 Jan 16;21(4):045708. Epub 2009 Dec 16.

Department of Physics, Tsinghua University, Beijing, People's Republic of China.

We report a simple and continuous spinning method that combines twisting and shrinking processes to produce carbon nanotube yarns. In this method, a yarn freshly spun from a super-aligned carbon nanotube array is first twisted and then passes through a volatile solvent for shrinking. The as-produced yarn consists of densely packed carbon nanotubes, and thus has a tensile strength up to about 1 GPa. The tensile strength depends on the diameter and the twisting angle of the yarn. Different kinds of solvents, such as water, ethanol, and acetone, are used to shrink the twisted yarns, and acetone shows the best shrinking effect. The origin of the solvent shrinking effect is investigated. Our method is favorable for continuous mass production of high strength carbon nanotube yarns with a wide range of diameters, especially ultra-thin yarns.
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http://dx.doi.org/10.1088/0957-4484/21/4/045708DOI Listing
January 2010

Controlled fabrication of high-quality carbon nanoscrolls from monolayer graphene.

Nano Lett 2009 Jul;9(7):2565-70

Department of Physics and Tsinghua-Foxconn Nanotechnology Research Center, Tsinghua University, Beijing 100084, China.

We report a simple and effective way of fabricating high-quality carbon nanoscrolls (CNSs), using isopropyl alcohol solution to roll up monolayer graphene predefined on SiO(2)/Si substrates. Transmission electron microscopy studies reveal that the CNS has a tube-like structure with a hollow core surrounded by graphene walls 0.35 nm apart. Raman spectroscopy studies show that the CNS is free of significant defects, and the electronic structure and phonon dispersion are slightly different from those of two-dimensional graphene. Finally, the CNS-based device is fabricated, directly on the SiO(2)/Si substrate. Electrical-transport measurements show that its resistance is weakly gate-dependent but strongly temperature-dependent. In addition, the CNS can sustain a high current density up to 5 x 10(7) A/cm(2), indicating that it is a good candidate for microcircuit interconnects. The controlled fabrication of high-quality CNSs may open up new opportunities for both fundamental and applied research of CNSs.
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http://dx.doi.org/10.1021/nl900677yDOI Listing
July 2009

Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels.

J Biol Chem 2008 Mar 3;283(10):6033-9. Epub 2008 Jan 3.

Departments of Internal Medicine and Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242, USA.

As a pathway for Na(+) reabsorption, the epithelial Na(+) channel ENaC is critical for Na(+) homeostasis and blood pressure control. Na(+) transport is regulated by Nedd4-2, an E3 ubiquitin ligase that decreases ENaC expression at the cell surface. To investigate the underlying mechanisms, we proteolytically cleaved/activated ENaC at the cell surface and then quantitated the rate of disappearance of cleaved channels using electrophysiological and biochemical assays. We found that cleaved ENaC channels were rapidly removed from the cell surface. Deletion or mutation of the Nedd4-2 binding motifs in alpha, beta, and gammaENaC dramatically reduced endocytosis, whereas a mutation that disrupts a YXXØ endocytosis motif had no effect. ENaC endocytosis was also decreased by silencing of Nedd4-2 and by expression of a dominant negative Nedd4-2 construct. Conversely, Nedd4-2 overexpression increased ENaC endocytosis in human embryonic kidney 293 cells but had no effect in Fischer rat thyroid epithelia. In addition to its effect on endocytosis, Nedd4-2 also increased the rate of degradation of the cell surface pool of cleaved alphaENaC. Together the data indicate that Nedd4-2 reduces ENaC surface expression by altering its trafficking at two distinct sites in the endocytic pathway, inducing endocytosis of cleaved channels and targeting them for degradation.
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http://dx.doi.org/10.1074/jbc.M708555200DOI Listing
March 2008

Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC.

J Biol Chem 2007 Jul 14;282(28):20207-12. Epub 2007 May 14.

Departments of Internal Medicine and Molecular Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242, USA.

Epithelial Na(+) absorption is regulated by Nedd4-2, an E3 ubiquitin-protein ligase that reduces expression of the epithelial Na(+) channel ENaC at the cell surface. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 binds to ENaC via PY motifs located in the C termini of alpha-, beta-, and gammaENaC. However, little is known about the mechanism by which Nedd4-2 regulates ENaC surface expression. Here we found that Nedd4-2 catalyzes ubiquitination of alpha-, beta-, and gammaENaC; Nedd4-2 overexpression increased ubiquitination, whereas Nedd4-2 silencing decreased ubiquitination. Although Nedd4-2 increased both mono/oligoubiquitinated and multiubiquitinated forms of ENaC, monoubiquitination was sufficient for Nedd4-2 to reduce ENaC surface expression and reduce ENaC current. Ubiquitination was disrupted by Liddle syndrome-associated mutations in ENaC or mutation of the catalytic HECT domain in Nedd4-2. Several findings suggest that the interaction between Nedd4-2 and ENaC is localized to the cell surface. First, Nedd4-2 bound to a population of ENaC at the cell surface. Second, Nedd4-2 catalyzed ubiquitination of cell surface ENaC. Third, Nedd4-2 selectively reduced ENaC expression at the cell surface but did not alter the quantity of immature ENaC in the biosynthetic pathway. Finally, Nedd4-2 induced degradation of the cell surface pool of ENaC. Together, the data suggest a model in which Nedd4-2 binds to and ubiquitinates ENaC at the cell surface, which targets surface ENaC for degradation, and thus, reduces epithelial Na(+) transport.
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http://dx.doi.org/10.1074/jbc.M611329200DOI Listing
July 2007

Liddle's syndrome mutations increase Na+ transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage.

Proc Natl Acad Sci U S A 2006 Feb 13;103(8):2805-8. Epub 2006 Feb 13.

Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242, USA.

Liddle's syndrome, an inherited form of hypertension, is caused by mutations that delete or disrupt a C-terminal PY motif in the epithelial Na+ channel (ENaC). Previous work indicates that these mutations increase expression of ENaC at the cell surface by disrupting its binding to Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. However, it remains uncertain whether this mechanism alone is responsible; increased activity of ENaC channels could also contribute to excessive Na+ transport in Liddle's syndrome. ENaC activity is controlled in part by its cleavage state; proteolytic cleavage produces channels with a high open-state probability, whereas uncleaved channels are inactive. Here, we found that Liddle's syndrome mutations have two distinct effects of ENaC surface expression, both of which contribute to increased Na+ transport. First, these mutations increased ENaC expression at the cell surface; second, they increased the fraction of ENaC at the cell surface that was cleaved (active). This disproportionate increase in cleavage was reproduced by expression of a dominant-negative Nedd4-2 or mutation of ENaC ubiquitination sites, interventions that disrupt ENaC endocytosis and lysosomal degradation. Conversely, overexpression of Nedd4-2 had the opposite effect, decreasing the fraction of cleaved ENaC at the cell surface. Thus, the data not only suggest that Nedd4-2 regulates epithelial Na+ transport in part by controlling the relative expression of cleaved and uncleaved ENaC at the cell surface but also provide a mechanism by which Liddle's syndrome mutations alter ENaC activity.
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http://dx.doi.org/10.1073/pnas.0511184103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1413842PMC
February 2006

Nedd4-2 phosphorylation induces serum and glucocorticoid-regulated kinase (SGK) ubiquitination and degradation.

J Biol Chem 2005 Feb 2;280(6):4518-23. Epub 2004 Dec 2.

Department of Internal Medicine and Physiology, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.

Serum and glucocorticoid-regulated kinase (SGK) plays a key role in the regulation of epithelial Na+ transport. SGK phosphorylates Nedd4-2, an E3 ubiquitin-protein ligase that targets the epithelial Na+ channel (ENaC) for degradation. Phosphorylation increases Na+ transport by reducing Nedd4-2 binding to ENaC, which increases ENaC expression at the cell surface. Thus, SGK expression must be tightly controlled to maintain Na+ homeostasis. This occurs in part by regulation of SGK transcription; a variety of signals including steroid hormones (aldosterone and glucocorticoids) increase SGK levels by inducing transcription of SGK. However, SGK has a short half-life, suggesting that SGK levels might also be controlled by regulation of SGK degradation. Here we found that SGK degradation is mediated in part by Nedd4-2. Consistent with this model, overexpression of Nedd4-2 decreased steady-state levels of SGK in a dose-dependent manner by increasing SGK ubiquitination and degradation in the 26S proteasome. Conversely, silencing of Nedd4-2 by RNA interference stabilized SGK. Nedd4-2 phosphorylation potentiates SGK degradation; degradation was reduced by Nedd4-2 and SGK mutations that disrupt phosphorylation or by inhibition of SGK kinase activity. Together with previous work, the data support a model in which SGK and Nedd4-2 regulate one another in a reciprocal manner. SGK phosphorylates Nedd4-2, which reduces Nedd4-2 binding and inhibition of ENaC. Conversely, phosphorylation increases Nedd4-2-mediated degradation of SGK. Thus, by phosphorylating Nedd4-2, SGK induces its own degradation. This feedback inhibition may fine-tune the regulation of epithelial Na+ absorption.
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http://dx.doi.org/10.1074/jbc.M411053200DOI Listing
February 2005

cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na(+) channel through convergent phosphorylation of Nedd4-2.

J Biol Chem 2004 Oct 24;279(44):45753-8. Epub 2004 Aug 24.

Department of Internal Medicine, and Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.

The epithelial Na(+) channel (ENaC) functions as a pathway for epithelial Na(+) transport, contributing to Na(+) homeostasis and blood pressure control. Vasopressin increases ENaC expression at the cell surface through a pathway that includes cAMP and cAMP-dependent protein kinase (PKA), but the mechanisms that link PKA to ENaC are unknown. Here we found that cAMP regulates Na(+) transport in part by inhibiting the function of Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. Consistent with this model, we found that cAMP inhibited Nedd4-2 by decreasing its binding to ENaC. Moreover, decreased Nedd4-2 expression (RNA interference) or overexpression of a dominant negative Nedd4-2 construct disrupted ENaC regulation by cAMP. Nedd4-2 was a substrate for phosphorylation by PKA in vitro and in cells; three Nedd4-2 residues were phosphorylated by PKA and were required for cAMP to inhibit Nedd4-2 (relative functional importance Ser-327 > Ser-221 > Thr-246). Previous work found that these residues are also phosphorylated by serum and glucocorticoid-inducible kinase (SGK), a downstream mediator by which aldosterone regulates epithelial Na(+) transport. Consistent with a functional interaction between these pathways, overexpression of SGK blunted ENaC stimulation by cAMP, whereas inhibition of SGK increased stimulation. Conversely, cAMP agonists decreased ENaC stimulation by SGK. The data suggest that cAMP regulates ENaC in part by phosphorylation and inhibition of Nedd4-2. Moreover, Nedd4-2 is a central convergence point for kinase regulation of Na(+) transport.
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http://dx.doi.org/10.1074/jbc.M407858200DOI Listing
October 2004

Bradycardia induces angiogenesis, increases coronary reserve, and preserves function of the postinfarcted heart.

Circulation 2004 Aug 9;110(7):796-802. Epub 2004 Aug 9.

Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA.

Background: We tested the hypothesis that induction of chronic bradycardia would trigger an upregulation of key growth factors and receptors, which would then lead to angiogenesis and improve coronary reserve in the left ventricle after myocardial infarction.

Methods And Results: Bradycardia was induced in rats by administering alinidine via osmotic pumps beginning 1 day after coronary artery ligation. Echocardiographic analysis was conducted before and after treatment. Morphometric analysis was used in perfusion-fixed hearts to document arteriolar and capillary growth. Western blots were used to evaluate growth factor and receptor changes. During the first week of treatment, vascular endothelial growth factor (VEGF), VEGF receptor 1 (Flt-1), and basic fibroblast growth factor proteins were higher in the treated group, whereas VEGF receptor 2 (Flk-1), angiopoietin-1, and angiopoietin-2 were not affected by treatment. After 3 weeks, VEGF protein remained elevated, and bradycardia was associated with a higher capillary length density in the border (40%) and remote (14%) regions and a higher arteriolar length density in the septum (62%), despite a greater increase in left ventricular mass. Although arteriolar length density increased in all size classes, the greatest increase occurred in the smallest (terminal) arterioles. This vascular growth was associated with a 23% greater coronary reserve. Echocardiography revealed a smaller increase in ventricular volume and a greater preservation of ejection fraction in rats treated with bradycardia.

Conclusions: Pharmacologic induction of bradycardia enhances vascularity and coronary reserve, preserves function of surviving myocardium, and therefore, is a noninvasive, therapeutic avenue that provides an alternative to gene therapy.
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http://dx.doi.org/10.1161/01.CIR.0000138933.85923.36DOI Listing
August 2004

DITPA stimulates arteriolar growth and modifies myocardial postinfarction remodeling.

Am J Physiol Heart Circ Physiol 2004 May;286(5):H1994-2000

Department of Anatomy and Cell Biology, University of Iowa, Iowa City, Iowa 52242, USA.

Myocardial infarction (MI) is characterized by ventricular remodeling, hypertrophy of the surviving myocardium, and an insufficient angiogenic response. Thyroxine is a powerful stimulus for myocardial angiogenesis. Male rats that underwent coronary artery ligation and subsequent MI were given 3,5-diiodothyropropionic acid (DITPA; MI+DITPA group) during a 3-wk period. We evaluated ventricular remodeling using echocardiography and histology and myocardial vessel growth using image analysis. Protein expression was assessed using Western blotting and immunohistochemistry. This study tested the hypothesis that the thyroxine analog DITPA facilitates angiogenesis and influences postinfarction remodeling in the surviving hypertrophic myocardium. The increase in the region of akinesis (infarct expansion) was blunted in the MI+DITPA rats compared with the MI group (3 vs. 21%); the treated rats had smaller percent increases in the left ventricular (LV) volume (64 +/- 14 vs. 95 +/- 12) and the LV volume-to-mass ratio (47 +/- 13 vs. 84 +/- 10) as well as a blunted decrease in ejection fraction (-9 +/- 8 vs. -30 +/- 7%). Arteriolar length density was higher after treatment in the largest (>50% of the free wall) infarcts (64 +/- 3 vs. 43 +/- 7). Angiogenic growth factors [vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)] and the angiopoietin receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Tie-2) values were elevated during the first week after infarction. DITPA did not cause additional increases in VEGF or Tie-2 values but did induce an increase in bFGF value after 3 days of treatment. This study provides the first evidence for an anatomical basis, i.e., attenuated ventricular remodeling and arteriolar growth, for improved function attributed to DITPA therapy of the infarcted heart. The favorable influences of DITPA on LV remodeling after large infarction are principally due to border zone preservation.
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http://dx.doi.org/10.1152/ajpheart.00991.2003DOI Listing
May 2004