Biomedical subjects

Hye-Won Shin

Publications and source records attributed to Hye-Won Shin.

At least 19 recordsLinked to original sources

PubMed2006

Airway nitric oxide release is reduced after PBS inhalation in asthma.

Exhaled nitric oxide (NO) is elevated in asthma, but the underlying mechanisms remain poorly understood. Recent results in subjects with asthma have reported a decrease in exhaled breath pH and ammonia, as well as altered expression and activity of glutaminase in both alveolar and airway epithelial cells. This suggests that pH-dependent nitrite conversion to NO may be a source of exhaled NO in the asthmatic airway epithelium. However, the anatomic location (i.e., airway or alveolar region) of this pH-dependent NO release has not been investigated and could impact potential therapeutic strategies. We quantified airway (proximal) and alveolar (peripheral) contributions to exhaled NO at baseline and then after PBS inhalation in stable (mild-intermittent to severe) asthmatic subjects (20-44 yr old; n = 9) and healthy controls (22-41 yr old; n = 6). The mean (SD) maximum airway wall flux (pl/s) and alveolar concentration (ppb) at baseline in asthma subjects and healthy controls was 2,530 (2,572) and 5.42 (7.31) and 1,703 (1,567) and 1.88 (1.29), respectively. Compared with baseline, there is a significant decrease in the airway wall flux of NO in asthma as early as 15 min and continuing for up to 60 min (maximum -28% at 45 min) after PBS inhalation without alteration of alveolar concentration. Healthy control subjects did not display any changes in exhaled NO. We conclude that elevated airway NO at baseline in asthma is reduced by inhaled PBS. Thus airway NO may be, in part, due to nitrite conversion to NO and is consistent with airway pH dysregulation in asthma.

Administration, Inhalation↗

PubMed2006

Structural basis for Rab11-dependent membrane recruitment of a family of Rab11-interacting protein 3 (FIP3)/Arfophilin-1.

Family of Rab11-interacting protein (FIP)3/Arfophlin-1 and FIP4/Arfophilin-2 are dual effectors for Rab11 and ADP ribosylation factor (ARF)5/ARF6, which are involved in membrane delivery from recycling endosomes to the plasma membrane during cytokinesis. Here, we define the distinct C-terminal binding regions of FIP3 and FIP4 for Rab11 and ARF5/ARF6. Furthermore, we determined the crystal structure of Rab11 in complex with the Rab11-binding domain (RBD) of FIP3. The long amphiphilic alpha-helix of FIP3-RBD forms a parallel coiled-coil homodimer, with two symmetric interfaces with two Rab11 molecules. The hydrophobic side of the RBD helix is involved in homodimerization and mediates the interaction with the Rab11 switch 1 region, whereas the opposite hydrophilic side interacts with the Rab11 switch 2 and is the major factor contributing to the binding specificity. The bivalent interaction of FIP3 with Rab11 at the C terminus allows FIP3 to coordinately function with other binding partners, including ARFs.

ADP-Ribosylation Factors↗

PubMed2006

A simple technique to characterize proximal and peripheral nitric oxide exchange using constant flow exhalations and an axial diffusion model.

The most common technique employed to describe pulmonary gas exchange of nitric oxide (NO) combines multiple constant flow exhalations with a two-compartment model (2CM) that neglects 1) the trumpet shape (increasing surface area per unit volume) of the airway tree and 2) gas phase axial diffusion of NO. However, recent evidence suggests that these features of the lungs are important determinants of NO exchange. The goal of this study is to present an algorithm that characterizes NO exchange using multiple constant flow exhalations and a model that considers the trumpet shape of the airway tree and axial diffusion (model TMAD). Solution of the diffusion equation for the TMAD for exhalation flows >100 ml/s can be reduced to the same linear relationship between the NO elimination rate and the flow; however, the interpretation of the slope and the intercept depend on the model. We tested the TMAD in healthy subjects (n = 8) using commonly used and easily performed exhalation flows (100, 150, 200, and 250 ml/s). Compared with the 2CM, estimates (mean +/- SD) from the TMAD for the maximum airway flux are statistically higher (J'aw(NO) = 770 +/- 470 compared with 440 +/- 270 pl/s), whereas estimates for the steady-state alveolar concentration are statistically lower (CA(NO) = 0.66 +/- 0.98 compared with 1.2 +/- 0.80 parts/billion). Furthermore, CA(NO) from the TMAD is not different from zero. We conclude that proximal (airways) NO production is larger than previously predicted with the 2CM and that peripheral (respiratory bronchioles and alveoli) NO is near zero in healthy subjects.

Adult↗

PubMed2006

Exercise-induced bronchoconstriction alters airway nitric oxide exchange in a pattern distinct from spirometry.

Exhaled nitric oxide (NO) is altered in asthmatic subjects with exercise-induced bronchoconstriction (EIB). However, the physiological interpretation of exhaled NO is limited because of its dependence on exhalation flow and the inability to distinguish completely proximal (large airway) from peripheral (small airway and alveolar) contributions. We estimated flow-independent NO exchange parameters that partition exhaled NO into proximal and peripheral contributions at baseline, postexercise challenge, and postbronchodilator administration in steroid-naive mild-intermittent asthmatic subjects with EIB (24-43 yr old, n = 9) and healthy controls (20-31 yr old, n = 9). The mean +/- SD maximum airway wall flux and airway diffusing capacity were elevated and forced expiratory flow, midexpiratory phase (FEF(25-75)), forced expiratory volume in 1 s (FEV(1)), and FEV(1)/forced vital capacity (FVC) were reduced at baseline in subjects with EIB compared with healthy controls, whereas the steady-state alveolar concentration of NO and FVC were not different. Compared with the response of healthy controls, exercise challenge significantly reduced FEV(1) (-23 +/- 15%), FEF(25-75) (-37 +/- 18%), FVC (-12 +/- 12%), FEV(1)/FVC (-13 +/- 8%), and maximum airway wall flux (-35 +/- 11%) relative to baseline in subjects with EIB, whereas bronchodilator administration only increased FEV(1) (+20 +/- 21%), FEF(25-75) (+56 +/- 41%), and FEV(1)/FVC (+13 +/- 9%). We conclude that mild-intermittent steroid-naive asthmatic subjects with EIB have altered airway NO exchange dynamics at baseline and after exercise challenge but that these changes occur by distinct mechanisms and are not correlated with alterations in spirometry.

Adaptation, Physiological↗

PubMed2006

AMY-1 (associate of Myc-1) localization to the trans-Golgi network through interacting with BIG2, a guanine-nucleotide exchange factor for ADP-ribosylation factors.

AMY-1 (associate of Myc-1) was originally identified as a c-Myc-binding protein that enhances the c-Myc transcription activity, and subsequently found to interact with A-kinase-anchoring proteins (AKAPs), including AKAP149, S-AKAP84 and AKAP95. We show here that, using anti-AMY-1 antibodies we raised, AMY-1 localizes to the trans-Golgi network (TGN) and the nucleus. To explore the possible function of AMY-1, we have undertaken a search for interacting partners by co-immunoprecipitation experiments using cells stably expressing FLAG-tagged AMY-1. Interestingly, we have found that AMY-1 interacts with BIG2 and BIG1, both of which are high molecular weight guanine-nucleotide exchange factors for ADP-ribosylation factors (ARFs) and mainly localize to the TGN. Furthermore, we have demonstrated that AMY-1 is associated with the TGN through interacting with BIG2 but not with BIG1 using an RNA interference approach, although AMY-1 can interact with both BIG1 and BIG2 in vitro. Taken together with the facts that BIG2 contains domains that bind to regulatory subunits of protein kinase A and that recruitment of ARF1 onto Golgi membranes is mediated, at least in part, by activation of protein kinase A, these results suggest that BIG2 alone or in concert with recruited AMY-1 coordinates ARF-mediated membrane trafficking and signaling pathways.

ADP-Ribosylation Factors↗

PubMed2005

Examining axial diffusion of nitric oxide in the lungs using heliox and breath hold.

Exhaled nitric oxide (NO) is highly dependent on exhalation flow; thus exchange dynamics of NO have been described by multicompartment models and a series of flow-independent parameters that describe airway and alveolar exchange. Because the flow-independent NO airway parameters characterize features of the airway tissue (e.g., wall concentration), they should also be independent of the physical properties of the insufflating gas. We measured the total mass of NO exhaled (A(I,II)) from the airways after five different breath-hold times (5-30 s) in healthy adults (21-38 yr, n = 9) using air and heliox as the insufflating gas, and then modeled A(I,II) as a function of breath-hold time to determine airway NO exchange parameters. Increasing breath-hold time results in an increase in A(I,II) for both air and heliox, but A(I,II) is reduced by a mean (SD) of 31% (SD 6) (P < 0.04) in the presence of heliox, independent of breath-hold time. However, mean (SD) values (air, heliox) for the airway wall diffusing capacity [3.70 (SD 4.18), 3.56 pl.s(-1).ppb(-1) (SD 3.20)], the airway wall concentration [1,439 (SD 487), 1,503 ppb (SD 644>)], and the maximum airway wall flux [4,156 (SD 2,502), 4,412 pl/s (SD 2,906)] using a single-path trumpet-shaped airway model that considers axial diffusion were independent of the insufflating gas (P > 0.55). We conclude that a single-path trumpet model that considers axial diffusion captures the essential features of airway wall NO exchange and confirm earlier reports that the airway wall concentration in healthy adults exceeds 1 ppm and thus approaches physiological concentrations capable of modulating smooth muscle tone.

Adult↗

PubMed2005

Roles of ARFRP1 (ADP-ribosylation factor-related protein 1) in post-Golgi membrane trafficking.

ADP-ribosylation factor (ARF)-related protein 1 (ARFRP1) is a small GTPase with significant similarity to the ARF family. However, little is known about the function of ARFRP1 in mammalian cells, although knockout mice of its gene are embryonic lethal. In the present study, we demonstrate that ARFRP1 is associated mainly with the trans-Golgi compartment and the trans-Golgi network (TGN) and is an essential regulatory factor for targeting of Arl1 and GRIP domain-containing proteins, golgin-97 and golgin-245, onto Golgi membranes. Furthermore, we show that, in concert with Arl1 and GRIP proteins, ARFRP1 is implicated in the Golgi-to-plasma membrane transport of the vesicular stomatitis virus G protein as well as in the retrograde transport of TGN38 and Shiga toxin from endosomes to the TGN.

ADP-Ribosylation Factors↗

PubMed2005

An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway.

Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kbeta, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinasebeta (PI3Kbeta), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis.

Animals↗

PubMed2005

Expression of BIG2 and analysis of its function in mammalian cells.

BIG2 is a member of brefeldin A-inhibited guanine nucleotide exchange factors for ADP-ribosylation factors (ARFs). Although BIG2 is associated mainly with the trans-Golgi network, we have recently revealed that some population of BIG2 is associated with the recycling endosome. Moreover, we have found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We have also demonstrated that the exchange activity of BIG2 is specific for class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF enhances the BIG2(E738K)-induced membrane tubulation. Therefore, we have proposed that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs. This article describes methods used for examining the function of BIG2 including basic protocols, which are not conventional because of cytotoxicity of BIG2 in Escherichia coli cells and its low expression efficiency in mammalian cells.

ADP-Ribosylation Factor 1↗

PubMed2005

Cellular and subcellular localization of EFA6C, a third member of the EFA6 family, in adult mouse Purkinje cells.

EFA6C is a third member of the EFA6 family of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factor 6 (ARF6). In this study, we first demonstrated that EFA6C indeed activated ARF6 more selectively than ARF1 by ARF pull-down assay. In situ hybridization histochemistry revealed that EFA6C mRNA was expressed predominantly in mature Purkinje cells and the epithelial cells of the choroid plexus in contrast to the ubiquitous expression of ARF6 mRNA throughout the brain. EFA6C mRNA was already detectable in the Purkinje cells at embryonic day 13, increased progressively during post-natal development and peaked during post-natal second week. In Purkinje cells, the immunoreactivity for EFA6C was localized particularly in the post-synaptic density as well as the plasma membranes of the cell somata, dendritic shafts and spines, while the immunoreactivity in their axon terminals in the deep cerebellar nuclei was very faint. These findings suggest that EFA6C may be involved in the regulation of the membrane dynamics of the somatodendritic compartments of Purkinje cells through the activation of ARF6.

ADP-Ribosylation Factor 6↗

PubMed2004

A new and more accurate technique to characterize airway nitric oxide using different breath-hold times.

Exhaled nitric oxide (NO) arises from both airway and alveolar regions of the lungs, which provides an opportunity to characterize region-specific inflammation. Current methodologies rely on vital capacity breathing maneuvers and controlled exhalation flow rates, which can be difficult to perform, especially for young children and individuals with compromised lung function. In addition, recent theoretical and experimental studies demonstrate that gas-phase axial diffusion of NO has a significant impact on the exhaled NO signal. We have developed a new technique to characterize airway NO, which requires a series of progressively increasing breath-hold times followed by exhalation of only the airway compartment. Using our new technique, we determined values (means +/- SE) in healthy adults (20-38 yr, n = 8) for the airway diffusing capacity [4.5 +/- 1.6 pl.s(-1).parts per billion (ppb)(-1)], the airway wall concentration (1,340 +/- 213 ppb), and the maximum airway wall flux (4,350 +/- 811 pl/s). The new technique is simple to perform, and application of this data to simpler models with cylindrical airways and no axial diffusion yields parameters consistent with previous methods. Inclusion of axial diffusion as well as an anatomically correct trumpet-shaped airway geometry results in significant loss of NO from the airways to the alveolar region, profoundly impacting airway NO characterization. In particular, the airway wall concentration is more than an order of magnitude larger than previous estimates in healthy adults and may approach concentrations (approximately 5 nM) that can influence physiological processes such as smooth muscle tone in disease states such as asthma.

Adult↗

PubMed2004

BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity.

Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is associated mainly with the trans-Golgi network. In the present study, we have revealed that another population of BIG2 is associated with the recycling endosome and found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We also have shown that BIG2 has an exchange activity toward class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF exaggerates the BIG2(E738K)-induced tubulation of endosomal membranes. These observations together indicate that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs.

ADP-Ribosylation Factors↗

PubMed2004

Probing the impact of axial diffusion on nitric oxide exchange dynamics with heliox.

Exhaled nitric oxide (NO) is a potential noninvasive index of lung inflammation and is thought to arise from the alveolar and airway regions of the lungs. A two-compartment model has been used to describe NO exchange; however, the model neglects axial diffusion of NO in the gas phase, and recent theoretical studies suggest that this may introduce significant error. We used heliox (80% helium, 20% oxygen) as the insufflating gas to probe the impact of axial diffusion (molecular diffusivity of NO is increased 2.3-fold relative to air) in healthy adults (21-38 yr old, n = 9). Heliox decreased the plateau concentration of exhaled NO by 45% (exhalation flow rate of 50 ml/s). In addition, the total mass of NO exhaled in phase I and II after a 20-s breath hold was reduced by 36%. A single-path trumpet model that considers axial diffusion predicts a 50% increase in the maximum airway flux of NO and a near-zero alveolar concentration (Ca(NO)) and source. Furthermore, when NO elimination is plotted vs. constant exhalation flow rate (range 50-500 ml/s), the slope has been previously interpreted as a nonzero Ca(NO) (range 1-5 ppb); however, the trumpet model predicts a positive slope of 0.4-2.1 ppb despite a zero Ca(NO) because of a diminishing impact of axial diffusion as flow rate increases. We conclude that axial diffusion leads to a significant backdiffusion of NO from the airways to the alveolar region that significantly impacts the partitioning of airway and alveolar contributions to exhaled NO.

Adult↗

PubMed2004

Characterizing airway and alveolar nitric oxide exchange during tidal breathing using a three-compartment model.

Exhaled nitric oxide (NO) may be a useful marker of lung inflammation, but the concentration is highly dependent on exhalation flow rate due to a significant airway source. Current methods for partitioning pulmonary NO gas exchange into airway and alveolar regions utilize multiple exhalation flow rates or a single-breath maneuver with a preexpiratory breath hold, which is cumbersome for children and individuals with compromised lung function. Analysis of tidal breathing data has the potential to overcome these limitations, while still identifying region-specific parameters. In six healthy adults, we utilized a three-compartment model (two airway compartments and one alveolar compartment) to identify two potential flow-independent parameters that represent the average volumetric airway flux (pl/s) and the time-averaged alveolar concentration (parts/billion). Significant background noise and distortion of the signal from the sampling system were compensated for by using a Gaussian wavelet filter and a series of convolution integrals. Mean values for average volumetric airway flux and time-averaged alveolar concentration were 2,500 +/- 2,700 pl/s and 3.2 +/- 3.4 parts/billion, respectively, and were strongly correlated with analogous parameters determined from vital capacity breathing maneuvers. Analysis of multiple tidal breaths significantly reduced the standard error of the parameter estimates relative to the single-breath technique. Our initial assessment demonstrates the potential of utilizing tidal breathing for noninvasive characterization of pulmonary NO exchange dynamics.

Adult↗

PubMed2004

Arousal with iv epinephrine depends on the depth of anesthesia.

PURPOSE: To investigate whether the depth of anesthesia affects the change in the bispectral index (BIS) caused by iv epinephrine during propofol anesthesia. METHODS: Forty women undergoing elective lower abdominal surgery received a propofol target controlled infusion (TCI) to maintain a modified Observer's Assessment of Alertness/Sedation (OAA/S) score of 2 (sedation period). Subsequently anesthesia was induced with propofol TCI 5 mug.mL(-1) and rocuronium 0.9 mg.kg(-1), and propofol continued so as to maintain general anesthesia at a BIS of 50 (general anesthesia period). Intravenous epinephrine at a dose of 10 mug.5 mL(-1) in normal saline (epinephrine group, n = 20) or normal saline 5 mL (control group, n = 20) was administered during both periods. The BIS, mean arterial pressure (MAP) and heart rate (HR) were measured immediately before, and one, two, three, four, six, eight, and ten minutes after injection. The modified OAA/S scale was evaluated during the sedation period. RESULTS: There was no significant change in the modified OAA/S scale, BIS, or hemodynamic variables compared to preinjection values during either sedation or general anesthesia in the control group. Intravenous epinephrine increased the BIS and modified OAA/S scale during sedation, but there was no increase in BIS during general anesthesia. Increases in HR and MAP were observed during both periods after iv epinephrine. CONCLUSION: Intravenous epinephrine 10 mug resulted in an arousal effect and an increase in BIS during sedation, but did not change the BIS during general anesthesia. These results suggest that the arousal effect of iv epinephrine during propofol anesthesia depends on anesthetic depth.

Adrenergic Agonists↗

PubMed2004

Dual control of membrane targeting by PtdIns(4)P and ARF.

In mammalian cells, three types of phosphatidylinositol 4-kinase (PI4K) are associated with the Golgi complex, where their product, phosphatidylinositol 4-phosphate [PtdIns(4)P], is concentrated. The role of PtdIns(4)P in this compartment and how the PtdIns(4)P-positive membrane domain is formed and maintained despite continuous membrane flow are, however, poorly understood. Recent work has shown that PtdIns(4)P and the small GTPase ARF1 function cooperatively in the recruitment of four-phosphate adaptor proteins (FAPPs) to the trans-Golgi network (TGN) and has implicated FAPPs in formation of the membrane domain and in post-Golgi trafficking.

1-Phosphatidylinositol 4-Kinase↗

PubMed2004

Guanine nucleotide-exchange factors for arf GTPases: their diverse functions in membrane traffic.

Small GTPases of the Arf family, by cycling between GDP-bound inactive and GTP-bound active states, play a crucial role not only in the regulation of membrane traffic and dynamics but also in rearrangement of actin cytoskeleton. The exchange of GDP for GTP on Arf is catalyzed by a family of guanine nucleotide-exchange factors (GEFs) containing a Sec7 domain. The Sec7 domain is a target of brefeldin A, which inhibits various trafficking processes and induces organelle disintegration. During the past few years, significant progress has been made in elucidating the structure and catalytic mechanism of the Sec7 domains and physiological functions of the Sec7 domain-containing Arf-GEFs. Here we review the structures and functions of Arf-GEFs by focusing on the regulation of membrane traffic.

ADP-Ribosylation Factors↗

PubMed2003

GAT (GGA and Tom1) domain responsible for ubiquitin binding and ubiquitination.

GGAs (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor (ARF)-binding proteins) are a family of monomeric adaptor proteins involved in membrane trafficking from the trans-Golgi network to endosomes. The GAT (GGA and Tom1) domains of GGAs have previously been shown to interact with GTP-bound ARF and to be crucial for membrane recruitment of GGAs. Here we show that the C-terminal subdomain of the GAT domain, which is distinct from the N-terminal GAT subdomain responsible for ARF binding, can bind ubiquitin. The binding is mediated by interactions between residues on one side of the alpha3 helix of the GAT domain and those on the so-called Ile-44 surface patch of ubiquitin. The binding of the GAT domain to ubiquitin can be enhanced by the presence of a GTP-bound form of ARF. Furthermore, GGA itself is ubiquitinated in a manner dependent on the GAT-ubiquitin interaction. These results delineate the molecular basis for the interaction between ubiquitin and GAT and suggest that GGA-mediated trafficking is regulated by the ubiquitin system as endosomal trafficking mediated by other ubiquitin-binding proteins.

Amino Acid Sequence↗