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1.  Direct Observation of Defects and Increased Ion Permeability of a Membrane Induced by Structurally Disordered Cu/Zn-Superoxide Dismutase Aggregates 
PLoS ONE  2011;6(12):e28982.
Interactions between protein aggregates and a cellular membrane have been strongly implicated in many protein conformational diseases. However, such interactions for the case of Cu/Zn superoxide dismutase (SOD1) protein, which is related to fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS), have not been explored yet. For the first time, we report the direct observation of defect formation and increased ion permeability of a membrane induced by SOD1 aggregates using a supported lipid bilayer and membrane patches of human embryonic kidney cells as model membranes. We observed that aggregated SOD1 significantly induced the formation of defects within lipid membranes and caused the perturbation of membrane permeability, based on surface plasmon resonance spectroscopy, atomic force microscopy and electrophysiology. In the case of apo SOD1 with an unfolded structure, we found that it bound to the lipid membrane surface and slightly perturbed membrane permeability, compared to other folded proteins (holo SOD1 and bovine serum albumin). The changes in membrane integrity and permeability were found to be strongly dependent on the type of proteins and the amount of aggregates present. We expect that the findings presented herein will advance our understanding of the pathway by which structurally disordered SOD1 aggregates exert toxicity in vivo.
PMCID: PMC3247219  PMID: 22216152
2.  Airway Smooth Muscle Sensitivity to Methacholine in Precision-Cut Lung Slices (PCLS) from Ovalbumin-induced Asthmatic Mice 
Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness (AHR) and reversible airway obstruction. Methacholine (MCh) is widely used in broncho-provocation test to evaluate airway resistance. For experimental investigation, ovalbumin-induced sensitization is frequently used in rodents (Ova-asthma). However, albeit the inflammatory histology and AHR in vivo, it remains unclear whether the MCh sensitivity of airway smooth muscle isolated from Ova-asthma is persistently changed. In this study, the contractions of airways in precision-cut lung slices (PCLS) from control, Ova-asthma, and IL-13 overexpressed transgenic mice (IL-13TG) were compared by analyzing the airway lumen space (AW). The airway resistance in vivo was measured using plethysmograph. AHR and increased inflammatory cells in BAL fluid were confirmed in Ova-asthma and IL-13TG mice. In the PCLS from all three groups, MCh concentration-dependent narrowing of airway lumen (ΔAW) was observed. In contrast to the AHR in vivo, the EC50 of MCh for ΔAW from Ova-asthma and IL-13TG were not different from control, indicating unchanged sensitivity to MCh. Although the AW recovery upon MCh-washout showed sluggish tendency in Ova-asthma, the change was also statistically insignificant. Membrane depolarization-induced ΔAW by 60 mM K+ (60K-contraction) was larger in IL-13TG than control, whereas 60K-contraction of Ova-asthma was unaffected. Furthermore, serotonin-induced ΔAW of Ova-asthma was smaller than control and IL-13TG. Taken together, the AHR in Ova-asthma and IL-13TG are not reflected in the contractility of isolated airways from PCLS. The AHR of the model animals seems to require intrinsic agonists or inflammatory microenvironment that is washable during tissue preparation.
PMCID: PMC4297764  PMID: 25605999
Airway; Asthma; Lung slice; Smooth muscle
3.  Disappearance of Hypoxic Pulmonary Vasoconstriction and O2-Sensitive Nonselective Cationic Current in Arterial Myocytes of Rats Under Ambient Hypoxia 
Acute hypoxia induces contraction of pulmonary artery (PA) to protect ventilation/perfusion mismatch in lungs. As for the cellular mechanism of hypoxic pulmonary vasoconstriction (HPV), hypoxic inhibition of voltage-gated K+ channel (Kv) in PA smooth muscle cell (PASMC) has been suggested. In addition, our recent study showed that thromboxane A2 (TXA2) and hypoxia-activated nonselective cation channel (INSC) is also essential for HPV. However, it is not well understood whether HPV is maintained in the animals exposed to ambient hypoxia for two days (2d-H). Specifically, the associated electrophysiological changes in PASMCs have not been studied. Here we investigate the effects of 2d-H on HPV in isolated ventilated/perfused lungs (V/P lungs) from rats. HPV was almost abolished without structural remodeling of PA in 2d-H rats, and the lost HPV was not recovered by Kv inhibitor, 4-aminopyridine. Patch clamp study showed that the hypoxic inhibition of Kv current in PASMC was similar between 2d-H and control. In contrast, hypoxia and TXA2-activated INSC was not observed in PASMCs of 2d-H. From above results, it is suggested that the decreased INSC might be the primary functional cause of HPV disappearance in the relatively early period (2 d) of hypoxia.
PMCID: PMC3823961  PMID: 24227949
Chronic hypoxia; Hypoxic pulmonary vasoconstriction; Nonselective cation channel; O2-sensitive ion channel; Pulmonary artery
4.  Wide Spectrum of Inhibitory Effects of Sertraline on Cardiac Ion Channels 
Sertraline is a commonly used antidepressant of the selective serotonin reuptake inhibitors (SSRIs) class. In these experiments, we have used the whole cell patch clamp technique to examine the effects of sertraline on the major cardiac ion channels expressed in HEK293 cells and the native voltage-gated Ca2+ channels in rat ventricular myocytes. According to the results, sertraline is a potent blocker of cardiac K+ channels, such as hERG, IKs and IK1. The rank order of inhibitory potency was hERG >IK1> IKs with IC50 values of 0.7, 10.5, and 15.2 µM, respectively. In addition to K+ channels, sertraline also inhibited INa and ICa, and the IC50 values are 6.1 and 2.6 µM, respectively. Modification of these ion channels by sertraline could induce changes of the cardiac action potential duration and QT interval, and might result in cardiac arrhythmia.
PMCID: PMC3484517  PMID: 23118556
Antidepressant; Cardiac; Ion channel; Selective serotonin reuptake inhibitor; Sertraline
5.  Requirement of Pretone by Thromboxane A2 for Hypoxic Pulmonary Vasoconstriction in Precision-cut Lung Slices of Rat 
Hypoxic pulmonary vasoconstriction (HPV) is physiologically important response for preventing mismatching between ventilation and perfusion in lungs. The HPV of isolated pulmonary arteries (HPV-PA) usually require a partial pretone by thromboxane agonist (U46619). Because the HPV of ventilated/perfused lungs (HPV-lung) can be triggered without pretone conditioning, we suspected that a putative tissue factor might be responsible for the pretone of HPV. Here we investigated whether HPV can be also observed in precision-cut lung slices (PCLS) from rats. The HPV in PCLS also required partial contraction by U46619. In addition, K+ channel blockers (4AP and TEA) required U46619-pretone to induce significant contraction of PA in PCLS. In contrast, the airways in PCLS showed reversible contraction in response to the K+ channel blockers without pretone conditioning. Also, the airways showed no hypoxic constriction but a relaxation under the partial pretone by U46619. The airways in PCLS showed reliable, concentration-dependent contraction by metacholine (EC50, ~210 nM). In summary, the HPV in PCLS is more similar to isolated PA than V/P lungs. The metacholine-induced constriction of bronchioles suggested that the PLCS might be also useful for studying airway physiology in situ.
PMCID: PMC3298827  PMID: 22416221
Lung slice; Hypoxic pulmonary vasoconstriction; Thromboxane A2; Airway smooth muscle
6.  Functional Expression of TRPV4 Cation Channels in Human Mast Cell Line (HMC-1) 
Mast cells are activated by specific allergens and also by various nonspecific stimuli, which might induce physical urticaria. This study investigated the functional expression of temperature sensitive transient receptor potential vanilloid (TRPV) subfamily in the human mast cell line (HMC-1) using whole-cell patch clamp techniques. The temperature of perfusate was raised from room temperature (RT, 23~25℃ to a moderately high temperature (MHT, 37~39℃ to activate TRPV3/4, a high temperature (HT, 44~46℃ to activate TRPV1, or a very high temperature (VHT, 53~55℃ to activate TRPV2. The membrane conductance of HMC-1 was increased by MHT and HT in about 50% (21 of 40) of the tested cells, and the I/V curves showed weak outward rectification. VHT-induced current was 10-fold larger than those induced by MHT and HT. The application of the TRPV4 activator 4α-phorbol 12,13-didecanoate (4αPDD, 1µM) induced weakly outward rectifying currents similar to those induced by MHT. However, the TRPV3 agonist camphor or TRPV1 agonist capsaicin had no effect. RT-PCR analysis of HMC-1 demonstrated the expression of TRPV4 as well as potent expression of TRPV2. The [Ca2+]c of HMC-1 cells was also increased by MHT or by 4αPDD. In summary, our present study indicates that HMC-1 cells express Ca2+-permeable TRPV4 channels in addition to the previously reported expression of TRPV2 with a higher threshold of activating temperature.
PMCID: PMC3034123  PMID: 21311684
Mast cell; TRPV cation channels; TRPV4 protein; Temperature; Non-selective cation channel; Human
7.  Inhibition of Hypoxic Pulmonary Vasoconstriction of Rats by Carbon Monoxide 
Journal of Korean Medical Science  2010;25(10):1411-1417.
Hypoxic pulmonary vasoconstriction (HPV), a unique response of pulmonary circulation, is critical to prevent hypoxemia under local hypoventilation. Hypoxic inhibition of K+ channel is known as an important O2-sensing mechanism in HPV. Carbon monoxide (CO) is suggested as a positive regulator of Ca2+-activated K+ channel (BKCa), a stimulator of guanylate cyclase, and an O2-mimetic agent in heme moiety-dependent O2 sensing mechanisms. Here we compared the effects of CO on the HPV (Po2, 3%) in isolated pulmonary artery (HPVPA) and in blood-perfused/ventilated lungs (HPVlung) of rats. A pretreatment with CO (3%) abolished the HPVPA in a reversible manner. The inhibition of HPVPA was completely reversed by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor. In contrast, the HPVlung was only partly decreased by CO. Moreover, the partial inhibition of HPVlung by CO was affected neither by the pretreatment with ODQ nor by NO synthase inhibitor (L-NAME). The CO-induced inhibitions of HPVPA and HPVlung were commonly unaffected by tetraethylammonium (TEA, 2 mM), a blocker of BKCa. As a whole, CO inhibits HPVPA via activating guanylate cyclase. The inconsistent effects of ODQ on HPVPA and HPVlung suggest that ODQ may lose its sGC inhibitory action when applied to the blood-containing perfusate.
PMCID: PMC2946648  PMID: 20890419
Anoxia; Pulmonary Artery; Carbon Monoxide; Guanylate Cyclase; Oxygen
8.  Inhibition of Arterial Myogenic Responses by a Mixed Aqueous Extract of Salvia Miltiorrhiza and Panax Notoginseng (PASEL) Showing Antihypertensive Effects 
The dried roots of Danshen (Salvia miltiorrhiza) and Sanchi (Panax notoginseng) have been widely used in traditional Chinese medicine for promoting blood circulation as well as various other bodily functions. Here we investigated the effects of a mixture of aqueous extracts of Danshen and Sanchi, named PASEL, on blood pressure and vascular contractility in rats. Orally administered PASEL (62.5 mg/kg and 250 mg/kg, for 5 weeks) lowered the blood pressure of spontaneous hypertensive rats (SHR) but this was not observed in normal Wistar-Kyoto rats (WKR). We then investigated the effects of PASEL on the arterial contraction of the small branches of cerebral arteries (CAs) and large conduit femoral arteries (FAs) in rats. PASEL did not affect high-K (KCl 60 mM)- or phenyleprine (PhE)-induced contracture of FAs. The myogenic response, a reactive arterial constriction in response to increased luminal pressure, of small CA was dose-dependently suppressed by PASEL in SHR as well as control rats. Interestingly, the KCl-induced contraction of small CAs was slowly reversed by PASEL, and this effect was more prominent in SHR than control WKR. PASEL did not inhibit angiotensin-converting enzyme (ACE) activity. These results demonstrated that the antihypertensive effect of PASEL might be primarily mediated by altering the arterial MR, not by direct inhibition of L-type Ca2+ channels or by ACE inhibition.
PMCID: PMC2766709  PMID: 19885012
Myogenic response; Herbal extract; Blood pressure; Hypertension
9.  Effects of Mixed Herbal Extracts from Parched Puerariae Radix, Gingered Magnoliae Cortex, Glycyrrhizae Radix and Euphorbiae Radix (KIOM-79) on Cardiac Ion Channels and Action Potentials 
Journal of Korean Medical Science  2009;24(3):403-412.
KIOM-79, a mixture of ethanol extracts from four herbs (parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhizae radix and Euphorbiae radix), has been developed for the potential therapeutic application to diabetic symptoms. Because screening of unexpected cardiac arrhythmia is compulsory for the new drug development, we investigated the effects of KIOM-79 on the action potential (AP) and various ion channel currents in cardiac myocytes. KIOM-79 decreased the upstroke velocity (Vmax) and plateau potential while slightly increased the duration of action potential (APD). Consistent with the decreased Vmax and plateau potential, the peak amplitude of Na+ current (INa) and Ca2+ current (ICa,L) were decreased by KIOM-79. KIOM-79 showed dual effects on hERG K+ current; increase of depolarization phase current (Idepol) and decreased tail current at repolarization phase (Itail). The increase of APD was suspected due to the decreased Itail. In computer simulation, the change of cardiac action potential could be well simulated based on the effects of KIOM-79 on various membrane currents. As a whole, the influence of KIOM-79 on cardiac ion channels are minor at concentrations effective for the diabetic models (0.1-10 µg/mL). The results suggest safety in terms of the risk of cardiac arrhythmia. Also, our study demonstrates the usefulness of the cardiac computer simulation in screening drug-induced long-QT syndrome.
PMCID: PMC2698184  PMID: 19543501
Heart; Action Potentials; Long-QT Syndrome; Herbal Extract; Ion Channels; hERG
10.  Acidic pH-activated Cl- Current and Intracellular Ca2+ Response in Human Keratinocytes 
The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and [Ca2+]c of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH (pHe≤ 5.5) activated outwardly rectifying Cl- current (ICl,pH) with slow kinetics of voltage-dependent activation. ICl,pH was potently inhibited by an anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS, 73.5% inhibition at 1 µM). ICl,pH became more sensitive to pHe by raising temperature from 24℃ to 37℃. HaCaT cells also expressed Ca2+-activated Cl- current (ICl,Ca), and the amplitude of ICl,Ca was increased by relatively weak acidic pHe (7.0 and 6.8). Interestingly, the acidic pHe (5.0) also induced a sharp increase in the intracellular [Ca2+] (Δ[Ca2+]acid) of HaCaT cells. The Δ[Ca2+]acid was independent of extracellular Ca2+, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed Δ[Ca2+]acid. In summary, we found ICl,pH and Δ[Ca2+]acid in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.
PMCID: PMC2788633  PMID: 19967053
Keratinocyte; Extracellular pH; Anion channel; Intracellular calcium; pH-activated Cl- current
11.  M3 Subtype of Muscarinic Receptors Mediate Ca2+ Release from Intracellular Stores in Rat Prostate Neuroendocrine Cells 
Journal of Korean Medical Science  2005;20(2):256-261.
Our previous studies document the expression of adrenoceptors and purinoceptors in the rat prostate neuroendocrine cells (RPNECs). However, a direct investigation of the receptors for acetylcholine (ACh) is still lacking in the prostate neuroendocrine cells. RPNECs were freshly isolated from the ventral lobes of rat prostate by using collagenase. Effects of ACh and various muscarinic antagonists on the intracellular Ca2+ concentration ([Ca2+]c) were investigated by using the fura-2 spectrofluorimetry. Single-cell RT-PCR analysis was applied to identify the transcripts for the muscarinic receptor subtypes. ACh (5 µM) induced a sharp transient increase in the [Ca2+]c of RPNECs, which was independent of the extracellular Ca2+. In the same RPNECs, high KCl (60 mM), phenylephrine (5 µM), UTP (P2Y1/2 agonist, 50 µM), and α,β-meATP (P2X1/3 agonist, 0.5 µM) also increased the [Ca2+]c. The ACh-induced [Ca2+]c change (Δ[Ca2+]c) was blocked by atropine or by para-fluorohexahydrosiladifenidol (M3 antagonist, 0.3 µM), but not by telenzepine (M1 antagonist, 1 µM) and himbacine (M2 and M4 antagonist, 1 µM). The single-cell RT-PCR demonstrated the selective expression of mRNAs for M3 in RPNECs. In summary, RPNECs express M3 muscarinic receptors that are linked to the release of Ca2+ from intracellular stores. The Ca2+ signals of RPNECs might mediate the parasympathetic regulation of prostate gland.
PMCID: PMC2808602  PMID: 15831997
Prostate; Neurosecretory Systems; Receptor, Muscarinic M3; Calcium Signaling; Rat; Fura-2
12.  Cross-Link Formation and Peptidoglycan Lattice Assembly in the FemA Mutant of Staphylococcus aureus 
Biochemistry  2014;53(9):1420-1427.
Staphylococcus aureus FemA mutant grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-13C]alanine, l-[3-13C]alanine, [2-13C]glycine, and l-[5-19F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between 13C-labeled alanines and 19F-labeled lysine incorporated in the peptidoglycan. The alanyl 13C labels were preselected for REDOR measurement by their proximity to the glycine label using 13C–13C spin diffusion. The observed 13C–13C and 13C–19F distances are consistent with a tightly packed, hybrid architecture containing both parallel and perpendicular stems in a repeating structural motif within the peptidoglycan.
PMCID: PMC3985804  PMID: 24517508
13.  Attenuation of Acetylcholine Activated Potassium Current (IKACh) by Simvastatin, Not Pravastatin in Mouse Atrial Cardiomyocyte: Possible Atrial Fibrillation Preventing Effects of Statin 
PLoS ONE  2014;9(10):e106570.
Statins, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, are associated with the prevention of atrial fibrillation (AF) by pleiotropic effects. Recent clinical trial studies have demonstrated conflicting results on anti-arrhythmia between lipophilic and hydrophilic statins. However, the underlying mechanisms responsible for anti-arrhythmogenic effects of statins are largely unexplored. In this study, we evaluated the different roles of lipophilic and hydrophilic statins (simvastatin and pravastatin, respectively) in acetylcholine (100 µM)-activated K+ current (IKACh, recorded by nystatin-perforated whole cell patch clamp technique) which are important for AF initiation and maintenance in mouse atrial cardiomyocytes. Our results showed that simvastatin (1–10 µM) inhibited both peak and quasi-steady-state IKACh in a dose-dependent manner. In contrast, pravastatin (10 µM) had no effect on IKACh. Supplementation of substrates for the synthesis of cholesterol (mevalonate, geranylgeranyl pyrophosphate or farnesyl pyrophosphate) did not reverse the effect of simvastatin on IKACh, suggesting a cholesterol-independent effect on IKACh. Furthermore, supplementation of phosphatidylinositol 4,5-bisphosphate, extracellular perfusion of phospholipase C inhibitor or a protein kinase C (PKC) inhibitor had no effect on the inhibitory activity of simvastatin on IKACh. Simvastatin also inhibits adenosine activated IKACh, however, simvastatin does not inhibit IKACh after activated by intracellular loading of GTP gamma S. Importantly, shortening of the action potential duration by acetylcholine was restored by simvastatin but not by pravastatin. Together, these findings demonstrate that lipophilic statins but not hydrophilic statins attenuate IKACh in atrial cardiomyocytes via a mechanism that is independent of cholesterol synthesis or PKC pathway, but may be via the blockade of acetylcholine binding site. Our results may provide important background information for the use of statins in patients with AF.
PMCID: PMC4199526  PMID: 25329899
14.  Staphylococcus aureus Peptidoglycan Stem Packing by Rotational-Echo Double Resonance NMR Spectroscopy 
Biochemistry  2013;52(21):10.1021/bi4005039.
Staphylococcus aureus grown in the presence of an alanine-racemase inhibitor was labeled with D-[1-13C]alanine and L-[15N]alanine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance NMR of intact whole cells was used to measure internuclear distances between 13C and 15N of labeled amino acids incorporated in the peptidoglycan, and from those labels to 19F of a glycopeptide drug specifically bound to the peptidoglycan. The observed 13C-15N average distance of 4.1 to 4.4 Å between D- and L-alanines in nearest-neighbor peptide stems is consistent with a local, tightly packed, parallel-stem architecture for a repeating structural motif within the peptidoglycan of S. aureus.
PMCID: PMC3796188  PMID: 23617832
alanine racemase; bacterial cell-wall tessera; glycopeptide antibiotic; REDOR; solid-state NMR
15.  Locations of the hydrophobic side chains of lipoglycopeptides bound to the peptidoglycan of Staphylococcus aureus§ 
Biochemistry  2013;52(20):10.1021/bi400054p.
Glycopeptides whose aminosugars have been modified by attachment of hydrophobic side chains are frequently active against vancomycin-resistant microorganisms. We have compared the conformations of six such fluorinated glycopeptides (with side chains of varying length) complexed to cell walls labeled with d-[1-13C]alanine, [1-13C]glycine, and l-[ε-15N]lysine in whole-cells of Staphylococcus aureus. The internuclear distances from 19F of the bound drug to the 13C and 15N labels of the peptidoglycan, and to the natural abundance 31P of lipid membranes and teichoic acids, were determined by rotational-echo double resonance NMR. The drugs did not dimerize, and their side chains did not form membrane anchors but instead became essential parts of secondary binding to pentaglycyl bridge segments of the cell-wall peptidoglycan.
PMCID: PMC3778154  PMID: 23607653
antibiotic; cell wall; oritavancin; REDOR; solid-state NMR; telavancin; vancomycin
16.  Isotridecanyl side chain of plusbacin-A3 is essential for the transglycosylase inhibition of peptidoglycan biosynthesis§ 
Biochemistry  2013;52(11):1973-1979.
Plusbacin-A3 (pb-A3) is a cyclic lipodepsipeptide which exhibits antibacterial activity against multidrug-resistant Gram-positive pathogens. Plusbacin-A3 is thought not to enter the cell cytoplasm and its lipophilic isotridecanyl side chain is presumed to insert into the membrane bilayer thereby facilitating either lipid II binding or some form of membrane disruption. Analogues of pb-A3, [2H]pb-A3 and deslipo-pb-A3, were synthesized to test membrane insertion as key to the mode of action. [2H]pb-A3 has a 2H-isotopically labeled isopropyl subunit of the lipid side chain, and deslipo-pb-A3 is missing the isotridecanyl side chain. Both analogues have the pb-A3 core structure. The loss of antimicrobial activity in deslipo-pb-A3 showed that the isotridecanyl side chain is crucial for the drug mode of action. However, rotational-echo double resonance NMR characterization of [2H]pb-A3 bound to [1-13C]glycine labeled whole-cells of Staphylococcus aureus showed that the isotridecanyl side chain does not insert into the lipid membrane, but instead is found in the staphylococcal cell wall, positioned near the pentaglycyl cross-bridge of the cell-wall peptidoglycan. Addition of [2H]pb-A3 during S. aureus growth resulted in an accumulation of Park’s nucleotide, consistent with the inhibition of the transglycosylation step of peptidoglycan biosynthesis.
PMCID: PMC3628776  PMID: 23421534
antibiotics; cell walls; cyclic peptides; REDOR; solid-state NMR
17.  Uniformity of Glycyl Bridge Lengths in the Mature Cell Walls of Fem Mutants of Methicillin-Resistant Staphylococcus aureus 
Journal of Bacteriology  2013;195(7):1421-1427.
Peptidoglycan (PG) composition in intact cells of methicillin-resistant Staphylococcus aureus (MRSA) and its isogenic Fem mutants has been characterized by measuring the glycine content of PG bridge structures by solid-state nuclear magnetic resonance (NMR). The glycine content estimated from integrated intensities (rather than peak heights) in the cell walls of whole cells was increased by approximately 30% for the FemA mutant and was reduced by 25% for the FemB mutant relative to expected values for homogeneous structures. In contrast, the expected compositions were observed in isolated cell walls of the same mutants. For FemA mutant whole cells, the increase was due to the presence of triglycyl bridge PG units (confirmed directly by mass spectrometric analysis), which constituted 10% of the total PG. These species were coalesced in some sort of a lattice or aggregate with spatial proximity to other PG bridges. This result suggests that the triglycyl-bridged PG units form a PG-like structure that is not incorporated into the mature cell wall.
PMCID: PMC3624537  PMID: 23335411
18.  Identification and Functional Characterization of Ion Channels in CD34+ Hematopoietic Stem Cells from Human Peripheral Blood 
Molecules and Cells  2011;32(2):181-188.
Hematopoietic stem cells (HSCs) are used therapeutically for hematological diseases and may also serve as a source for nonhematopoietic tissue engineering in the future. In other cell types, ion channels have been investigated as potential targets for the regulation of proliferation and differentiation. However, the ion channels of HSCs remain elusive. Here, we functionally characterized the ion channels of CD34+ cells from human peripheral blood. Using fluorescence-activated cell sorting, we confirmed that the CD34+ cells also express CD45 and CD133. In the CD34+/ CD45+/CD133high HSCs, RT-PCR of 58 ion channel mRNAs revealed the coexpression of Kv1.3, Kv7.1, Nav1.7, TASK2, TALK2, TWIK2, TRPC4, TRPC6, TRPM2, TRPM7, and TRPV2. Whole-cell patch clamp recordings identified voltage-gated K+ currents (putatively Kv1.3), pH-sensitive TASK2-like background K+ currents, ADP-ribose-activated TRPM2 currents, temperature-sensitive TRPV2-like currents, and diacylglycerol- analogue-activated TRPC6-like currents. Our results lend new insight into the physiological role of ion channels in HSCs, the specific implications of which require further investigation.
PMCID: PMC3887668  PMID: 21638203
CD34; hematopoietic stem cell; ion channel; potassium channel; TRP channel
19.  CD40 Co-stimulation Inhibits Sustained BCR-induced Ca2+ Signaling in Response to Long-term Antigenic Stimulation of Immature B Cells 
Regulation of B cell receptor (BCR)-induced Ca2+ signaling by CD40 co-stimulation was compared in long-term BCR-stimulated immature (WEHI-231) and mature (Bal-17) B cells. In response to long-term pre-stimulation of immature WEHI-231 cells to α-IgM antibody (0.5~48 hr), the initial transient decrease in BCR-induced [Ca2+]i was followed by spontaneous recovery to control level within 24 hr. The recovery of Ca2+ signaling in WEHI-231 cells was not due to restoration of internalized receptor but instead to an increase in the levels of PLCγ2 and IP3R-3. CD40 co-stimulation of WEHI-231 cells prevented BCR-induced cell cycle arrest and apoptosis, and it strongly inhibited the recovery of BCR-induced Ca2+ signaling. CD40 co-stimulation also enhanced BCR internalization and reduced expression of PLCγ2 and IP3R-3. Pre-treatment of WEHI-231 cells with the antioxidant N-acetyl-L-cysteine (NAC) strongly inhibited CD40-mediated prevention of the recovery of Ca2+ signaling. In contrast to immature WEHI-231 cells, identical long-term α-IgM pre-stimulation of mature Bal-17 cells abolished the increase in BCR-induced [Ca2+]i, regardless of CD40 co-stimulation. These results suggest that CD40-mediated signaling prevents antigen-induced cell cycle arrest and apoptosis of immature B cells through inhibition of sustained BCR-induced Ca2+ signaling.
PMCID: PMC3154383  PMID: 21860597
B cell receptor; Ca2+; CD40; Reactive oxygen species; WEHI-231
20.  Suppression of CFTR-mediated Cl- Secretion of Airway Epithelium in Vitamin C-deficient Mice 
Journal of Korean Medical Science  2011;26(3):317-324.
Hyperoxic ventilation induces detrimental effects on the respiratory system, and ambient oxygen may be harmful unless compensated by physiological anti-oxidants, such as vitamin C. Here we investigate the changes in electrolyte transport of airway epithelium in mice exposed to normobaric hyperoxia and in gulonolacton oxidase knock-out (gulo[-/-]) mice without vitamin C (Vit-C) supplementation. Short-circuit current (Isc) of tracheal epithelium was measured using Ussing chamber technique. After confirming amiloride-sensitive Na+ absorption (ΔIsc,amil), cAMP-dependent Cl- secretion (ΔIsc,forsk) was induced by forskolin. To evaluate Ca2+-dependent Cl- secretion, ATP was applied to the luminal side (ΔIsc,ATP). In mice exposed to 98% PO2 for 36 hr, ΔIsc,forsk decreased, ΔIsc,amil and ΔIsc,ATP was not affected. In gulo(-/-) mice, both ΔIsc,forsk and ΔIsc,ATP decreased from three weeks after Vit-C deprivation, while both were unchanged with Vit-C supplementation. At the fourth week, tissue resistance and all electrolyte transport activities were decreased. An immunofluorescence study showed that the expression of cystic fibrosis conductance regulator (CFTR) was decreased in gulo(-/-) mice, whereas the expression of KCNQ1 K+ channel was preserved. Taken together, the CFTR-mediated Cl- secretion of airway epithelium is susceptible to oxidative stress, which suggests that supplementation of the antioxidant might be beneficial for the maintenance of airway surface liquid.
PMCID: PMC3051076  PMID: 21394297
Hyperoxia; Airway Epithelium; Cystic Fibrosis Transmembrane Conductance Regulator; Electrolyte Transport; Ascorbic Acid
21.  Vancomycin and Oritavancin Have Different Modes of Action in Enterococcus faecium 
Journal of molecular biology  2009;392(5):1178-1191.
The increasing frequency of Enterococcus faecium isolates with multidrug resistance is a serious clinical problem given the severely limited number of therapeutic options available to treat these infections. Oritavancin is a promising new alternative in clinical development that has potent antimicrobial activity against both staphylococcal and enterococcal vancomycin-resistant pathogens. Using solid-state NMR to detect changes in the cell-wall structure and peptidoglycan precursors of whole cells after antibiotic-induced stress, we report that vancomycin and oritavancin have different modes of action in E. faecium. Our results show the accumulation of peptidoglycan precursors after vancomycin treatment, consistent with transglycosylase inhibition, but no measurable difference in cross-linking. In contrast, after oritavancin exposure, we do not observe the accumulation of peptidoglycan precursors. Instead, the number of cross-links is significantly reduced, showing that oritavancin primarily inhibits transpeptidation. We propose that the activity of oritavancin is the result of a secondary-binding interaction with the E. faecium peptidoglycan. The hypothesis is supported by results from 13C{19F} REDOR experiments on whole cells enriched with L-[1-13C]lysine and complexed with desleucyl [19F]oritavancin. These experiments establish that an oritavancin derivative with a damaged D-Ala-D-Ala binding pocket still binds to E. faecium peptidoglycan. The 13C{19F} REDOR dephasing maximum indicates that the secondary-binding site of oritavancin is specific to nascent and template peptidoglycan. We conclude that the inhibition of transpeptidation by oritavancin in E. faecium is the result of the large number of secondary-binding sites relative to the number of primary-binding sites.
PMCID: PMC2748155  PMID: 19576226
bacterial cell wall; glycopeptide antibiotics; peptidoglycan; REDOR; solid-state NMR
22.  Oritavancin Binds to Isolated Protoplast Membranes but not Intact Protoplasts of Staphylococcus aureus 
Journal of molecular biology  2009;391(2):414-425.
Solid-state NMR has been used to examine the binding of [19F]oritavancin, a fluorinated analogue of oritavancin, to isolated protoplast membranes and whole-cell sucrose-stabilized protoplasts of Staphylococcus aureus, grown in media containing [1-13C]glycine and L-[ε-15N]lysine. Rotational-echo double-resonance NMR was used to characterize the binding by estimating internuclear distances from 19F of the oritavancin to 13C and 15N labels of the membrane-associated peptidoglycan, and to the 31P of the phospholipid bilayer of the membrane. In isolated protoplast membranes, both with and without 1M sucrose added to the buffer, the nascent peptidoglycan was extended away from the membrane surface and the oritavancin hydrophobic sidechain was buried deep in the exposed lipid bilayer. However, there was no [19F]oritavancin binding to intact sucrose-stabilized protoplasts, even though the drug bound normally to the cell walls of whole cells of S. aureus in the presence of 1M sucrose. As shown by the proximity of peptidoglycan-bridge 13C labels to phosphate 31P, the nascent peptidoglycan of the intact protoplasts was confined to the membrane surface.
PMCID: PMC2747642  PMID: 19538971
Bacterial cell wall; glycopeptide antibiotic; rotational-echo double resonance; solid-state NMR; transglycosylase
23.  The Effect of Brimonidine on Transepithelial Resistance in a Human Retinal Pigment Epithelial Cell Line 
To investigate the effects of brimonidine, an α-2-adrenergic agonist, on barrier function in ARPE-19 cells by measuring transepithelial resistance (TER).
ARPE-19 cells were cultured into a confluent monolayer on a microporous filter. Brimonidine was added to the apical medium, and the barrier function of the cells was evaluated by measuring TER. A subset of cells was treated under hypoxic conditions, and the TER changes observed upon administration of brimonidine were compared to those observed in cells in normoxic conditions.
The ARPE cell membrane reached a peak resistance of 29.1±7.97 Ωcm2 after four weeks of culture. The TER of the cells treated under normoxic conditions increased with brimonidine treatment; however, the TER of the cells treated under hypoxic conditions did not change following the administration of brimonidine.
Barrier function in ARPE-19 cells increased with brimonidine treatment. Understanding the exact mechanism of this barrier function change requires further investigation.
PMCID: PMC2882081  PMID: 20532144
Adrenergic agonist; Blood-retinal barrier; Brimonidine; Retinal pigment epithelium
24.  Characterization of Peptidoglycan in Fem-deletion Mutants of Methicillin-resistant Staphylococcus aureus by Solid-State NMR† 
Biochemistry  2009;48(14):3100-3108.
Compositional analysis of the peptidoglycan (PG) of a wild-type methicilin-resistant Staphylococcus aureus and its fem-deletion mutants has been performed on whole cells and cell walls using stable-isotope labeling and rotational-echo double-resonance NMR. The labels included [1-13C, 15N]glycine and L-[ε-15N]lysine (for a direct measure of the number of glycyl residues in the bridging segment), [1-13C]glycine and L-[ε-15N]lysine (concentration of bridge-links), and D-[1-13C]alanine and [15N]glycine (concentrations of cross-links and wall teichoic acids). The bridging segment length changed from 5.0 glycyl residues (wild-type strain) to 2.5±0.1 (FemB) with modest changes in cross-link and bridge-link concentrations. This accurate in situ measurement for the FemB mutant indicates a heterogeneous PG-structure with 25% monoglycyl- and 75% triglycyl-bridges. When the bridging segment was reduced to a single (1.0±0.1) glycyl residue (FemA), cross-linking decreased by more than 20%, resulting in a high concentration of open N-terminus glycyl segments.
PMCID: PMC2785074  PMID: 19309106
Fem factors; magic-angle spinning; rotational-echo double resonance; transglycosylase; transpeptidase
25.  Staphylococcus aureus Peptidoglycan Tertiary Structure from Carbon-13 Spin Diffusion 
The cell-wall peptidoglycan of Staphylococcus aureus is a heterogeneous, highly cross-linked polymer of unknown tertiary structure. We have partially characterized this structure by measuring spin diffusion from 13C labels in pentaglycyl cross-linking segments to natural-abundance 13C in the surrounding intact cell walls. The measurements were performed using a version of centerband-only detection of exchange (CODEX). The cell walls were isolated from S. aureus grown in media containing [1-13C]glycine. The CODEX spin diffusion rates established that the pentaglycyl bridge of one peptidoglycan repeat unit of S. aureus is within 5 Å of the glycan chain of another repeat unit. This surprising proximity is interpreted in terms of a model for the peptidoglycan lattice in which all peptide stems in a plane perpendicular to the glycan mainchain are parallel to one another.
PMCID: PMC2778264  PMID: 19419167

Results 1-25 (31)