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91.
Development of an opinion leader-led HIV prevention intervention among alcohol users in Chennai, India. 总被引:1,自引:0,他引:1
Sudha Sivaram Aylur K Srikrishnan Carl A Latkin Sethulakshmi C Johnson Vivian F Go Margaret E Bentley Suniti Solomon David D Celentano 《AIDS education and prevention》2004,16(2):137-149
In 1999, we began a community-based randomized controlled prevention trial in Chennai, which aims to test the efficacy of HIV prevention messages disseminated through members of an individual's social group called community popular opinion leaders, or CPOLs. We targeted patrons of 100 bars or wine shops in the city of Chennai, India. In this article we report on the process of development of an HIV prevention intervention for wine shop patrons. First, we conducted detailed ethnography to understand social norms and CPOL and social network characteristics, including 41 in-depth interviews among wine shop patrons and gatekeepers. Second, we tailored a generic HIV education training manual to appropriately address the needs of Chennai wine shop patrons. Field-testing involved 16 focus groups with wine shop patrons and 12 sessions of participant observations in wine shops. Finally, we piloted the intervention to determine the appropriateness of the training program and its content among wine shop patrons. Our ethnographic data indicated that wine shops are a common meeting place for men. We were able to identify CPOLs influential in these settings and train them to deliver appropriate prevention messages to their close friends and associates. We found that HIV prevention messages in this population need to dispel misperceptions about HIV transmission, provide strategies and skills to adopt and sustain condom use, and target the role of alcohol in sexual behavior. We outline specific lessons we learned in intervention development in this population. 相似文献
92.
We have developed hybridization-sensitive fluorescent oligonucleotide probes that, in the presence of quencher strands, undergo efficient fluorescence quenching through the formation of partial DNA/DNA duplexes. In the presence of target RNA, rapid displacement of the quencher strands results in highly enhanced fluorescence.We have developed hybridization-sensitive fluorescent oligonucleotide probes that, in the presence of quencher strands, undergo efficient fluorescence quenching through the formation of partial DNA/DNA duplexes.The detection of biomolecules is an important part of any investigation into their biological mechanisms and phenomena. Fluorescence-based methods are particularly useful for providing interpretable signals for various targets (e.g., genes, proteins, small molecules). When nucleic acids are used as probes, they can provide sequence-specific information regarding the binding (through hydrogen bonding) of target nucleic acids. Because of their high sequence-specificity, many fluorescent hybridization probes, including molecular beacons (MBs), have been developed and applied for nucleic acid detection and visualization.1,2In previous studies, we found that a quencher-free molecular beacon (QF-MB) containing the pyrene-modified nucleoside PyU exhibited a high fluorescence enhancement in the presence of trinucleotide repeats, especially for RNA.3,4 Among various fluorescent nucleobase derivatives, uracil derivatives have been particularly useful for selective detection of specific sequences, taking advantage of changes in photoinduced electron transfer between the fluorophore and the neighboring base.3–5 To apply such systems to various other target sequences, here we designed fully complementary sequences and incorporated the internal fluorescent nucleoside PyU in place of a thymine residue, resulting in a significantly increased fluorescence signal based on strand displacement (Fig. 1). Incorporation of a PyU unit in a single strand of the probe sequence and hybridization with a strand partially complementary to the probe strand containing a pyrene unit as a fluorescence quencher can lead to improved discrimination factors.4,6 Such partially double-stranded probes have several attractive features.Open in a separate windowFig. 1(A) Schematic representation of the strand displacement process developed in this present system. (B) Structures of the internal fluorophore PyU.First, the probe sequence does not require an additional sequence in its strand that is not complementary to the target sequence to ensure formation of a secondary structure (e.g., a hairpin). Such additional sequences might interfere with the specific hybridization between the probe and the target sequence. Accordingly, double-stranded fluorescence probes should allow the specific detection of many kinds of targets. Second, the highly quenched initial fluorescence signal, due to the formation of a partial duplex, results in significantly increased fluorescence in the presence of the target; the incorporation of a hybridization-sensitive internal fluorophore provides a stable and sensitive fluorescence signal upon perfect hybridization with the target.Cofilin is a protein that regulates the activity of actin, which is related to the formation of the cytoskeleton in cells. Actin plays a crucial role in the growth and elongation of cells,7 especially in neurons and, therefore, in the control of neurotransmission. We designed probe strands complementary to the 3′-untranslated region (3′-UTR) of target cofilin mRNA and synthesized three kinds of 19-mer probe strands (P1–P3) containing one or two PyU units in each strand (Fig. 2A and S1, ESI†). The fluorescence intensities of P1 and P3 increased dramatically after binding with the target RNA T19—by 17.6- and 16.0-fold, respectively. For the probe P2 (in which the PyU residue was located close to the 3′-end), however, the fluorescence enhancement was very low: only 1.8-fold (Table S2, ESI†). We assume that terminal modification of the PyU unit in the probe resulted in weak base pairs around the PyU residue than did central modification, resulting in a decrease in fluorescence enhancement (Table S3, ESI†).8 Because flanking base pairs around the PyU unit are relatively less rigid compared to central base pairs, the microenvironment of PyU in the duplex formed from P2 and T19 is different from the central modification. Therefore, the fluorescence intensity did not increase significantly compared to the single-stranded P2. The absorption spectrum of P2 in the presence of T19 exhibited a relatively less intense absorption band than that of the duplex formed from P1 and T19—the latter featured an intense signal corresponding to the high fluorescence intensity (Fig. S1, ESI†).9,10 Even though two PyU units were incorporated into the single strand P3, its fluorescence enhancement was similar to that of probe P1 containing only one PyU unit. Among other examined probe sequences complementary to other parts of 3′-UTR in cofilin mRNA, P6 (containing two PyU units) also exhibited fluorescence intensity similar to those of P4 and P5, single-PyU – containing probes each modified in the central position (Fig. S2, ESI†). These results suggest that single modification of a PyU unit in the probe sequence is more efficient than dual modification, in terms of inducing high fluorescence enhancement with target RNA.Oligonucleotide sequences of probe and target strands
Open in a separate windowaTarget RNA sequence.bUnderlined letter indicates a single mismatched base.Open in a separate windowFig. 2(A, B) Fluorescence emission spectra of (A) the probes P1–P3 with T19 and (B) the probe P1 with U5 and U6. (C) Fluorescence enhancements (F/F0) at 432 nm of P1 in the presence of U5 and U6 upon binding with T19; 1.0 μM of samples in 100 mM Tris-HCl buffer (pH 7.2), 100 mM NaCl and 10 mM MgCl2; annealing: 90 °C; excitation wavelength: 380 nm; excitation/emission slit: 5 nm/5 nm; F: fluorescence intensity at 432 nm of the probe P1 with the target RNA T19 in the absence or presence of a quencher strand U5 and U6; F0: fluorescence intensity at 432 nm of the probe P1 in the absence or presence of a quencher strand U5 and U6.Next, to improve the fluorescence enhancement in the presence of the target, the background signal of the probe was decreased by mixing it with a pyrene-modified short oligonucleotide, a so-called “quencher strand”, capable of quenching the fluorescence of the PyU unit. Two pyrene units on the opposite side in the duplex resulted in fluorescence quenching because pyrene moieties are stacked each other and located in a highly polar environment.4,9 Such duplexes containing probe and quencher strands would have to undergo displacement of the quencher strand prior to hybridization of the target strand. First, we tested the effects of a PyU residue located in the quencher strand at the central position, opposite the PyU residue in the probe strand, potentially minimizing the fluorescence of the PyU residue in the probe strand through π-stacking of the two pyrene units in the duplex.4,11 We synthesized the quencher strands U5 and U6, each containing a PyU residue, and examined the relationship between the quenching efficiency and stable hybridization of the probe/quencher duplexes upon varying the length of the quencher strand (Fig. 2B). The more stable the partial duplex is formed, the more effective stacking interaction between pyrene moieties close to each other can be possible.12 In the presence of the PyU-modified quencher strands, the quenching efficiency of the probe at 435 nm was 65% for U5 and 80% for U6. As a result, the enhancements in fluorescence for the probe in the presence of the target T19 were 51.5- and 66.7-fold for U5 and U6, respectively (Fig. 2C and S7, ESI†) much higher than that for P1 alone. Notably, these fluorescence signals were generated not only from the probe/target duplexes but also from the released quencher strands (i.e., the single-stranded quencher strands also exhibited fluorescence to some degree). Therefore, the actual fluorescence signal arising from hybridization of the probe with the target was slightly lower than that observed in the fluorescence spectra; we estimated that the additional signals due to the release of U5 and U6 increased the fluorescence intensity by 7% (Fig. S7, ESI†). In other words, the released quencher strands added to the fluorescence enhancement of the DNA/RNA duplex. Moreover, we also tested the effect of incorporating a dabcyl derivative, DabU, as a typical fluorescence quencher on the quencher strand and compared its effects with those of the quencher strand containing a PyU unit (Table S4, Fig. S4, ESI†). The PyU-modified quencher strands provided the probe with similar quenching in fluorescence as did DabU-modified quencher strands of the same length (Fig. S8 and S9, ESI†). The melting temperatures (Fig. S10, Table S5, ESI†) of the duplexes of the PyU-modified quencher strands and P1 (for U5 and U6 with P1: 60.1 and 66.0 °C, respectively) were higher than those of the natural strands and P1 (for N5 and N6 with P1: 46.8 and 56.0 °C, respectively); the former were stabilized through π-stacking of the PyU units (Fig. S5 and S6, ESI†). The formation of duplexes from the probe and quencher strands was evident also in circular dichroism (CD) and polyacrylamide gel electrophoresis (PAGE) experiments (Fig. S11 and S12, ESI†).To confirm the effective strand displacement of the quencher strand from the probe strand, we conducted time-dependent fluorescence experiments after addition of the target RNA T19 to probe/quencher duplexes (Fig. 3). After addition of the target strand to the single strand of P1, hybridization was complete within 30 min (i.e., the increase in fluorescence at 435 nm was minor thereafter). In contrast, the fluorescence intensity of P1 in the presence of the 11-mer quencher strand U5 was relatively rapid, reaching equilibrium after 20 min; for the 13-mer strand U6, however, equilibrium was reached within 35 min. Thus, compared with the single-stranded probe P1, the hybrid of P1 with U5 reacted more rapidly with the target T19, but the reaction time of the hybrid of P1 with U6 responding to the target T19 was slightly slower than that of P1 in the absence of a quencher strand. We suspect that the probe strand in the absence of a quencher strand was stabilized by stacking of the nucleobases; the probe would take some time to hybridize with the target RNA, requiring unfolding of its stacked bases. For the partially hybridized duplexes, however, the non-bonded sequence of the probe would be exposed, facilitating hybridization with the target strand. As a result, the response of P1 in the presence of U5 toward the target RNA was slightly faster than that of single-stranded P1 alone. Thus, as the length of the non-bonding sequence of P1 in the partial duplex decreased by increasing the length of quencher strand, the rate of strand displacement decreased accordingly.13–16 Indeed, the reaction time for the probe strand in the presence of the 15-mer strand U7 was much longer than those in the presence of the 11- and 13-mer quencher strands, because only a four-nucleotide sequence was available for hybridization of the target stand T19 (Fig. S13, ESI†); in addition, the equilibrium of the reaction shifted to the left, in conjunction with a smaller enhancement in fluorescence.Open in a separate windowFig. 3Time-dependent fluorescence of P1 in the presence of U5 and U6 after addition of T19; 1.0 μM of samples in 100 mM Tris-HCl buffer (pH 7.2), 100 mM NaCl and 10 mM MgCl2; excitation wavelength: 380 nm; emission wavelength: 435 nm; excitation/emission slit: 5 nm/5 nm; temperature: 20 °C.We also tested the selectivity of the probe P1 itself against single-base-mismatched target RNA (Fig. 4, Open in a separate windowFig. 4Fluorescence emission spectra of P1 in the presence of single-base-mismatched target RNA; 1.0 μM of sample in 100 mM Tris-HCl buffer (pH 7.2), 100 mM NaCl and 10 mM MgCl2; annealing: 90 °C; excitation wavelength: 380 nm; excitation/emission slit: 5 nm/5 nm.In conclusion, we have developed a double-stranded duplex that functions as a universal probe that is highly specific for the sequence of its target RNA—in this case, for cofilin mRNA. When the PyU-modified probe strand was partially hybridized with quencher strands containing a PyU unit, the fluorescence intensity decreased dramatically as a result of π-stacking of the PyU units. The probe/quencher hybrids provided even greater fluorescence enhancements after stable binding of the target RNA strand with the additional signal from the released quencher strand further improving the fluorescence detection of the target RNA. 相似文献
Name | Sequence |
---|---|
P1 | 5′-GGT GCC PyUAG GAC GGG ACT T-3′ |
P2 | 5′-GGT GCC TAG GAC GGG ACPyU T-3′ |
P3 | 5′-GGT GCC PyUAG GAC GGG ACPyU T-3′ |
U5 | 3′-CA CGG PyUTC CTG-5′ |
U6 | 3′-CCA CGG PyUTC CTG C-5′ |
T19a | 5′-a agu ccc guc cua ggc acc-3′ |
T19-Ua,b | 5′-a agu ccc guc cuu̲ ggc acc-3′ |
T19-Ga,b | 5′-a agu ccc guc cug̲ ggc acc-3′ |
T19-Ca,b | 5′-a agu ccc guc cuc̲ ggc acc-3′ |
93.
Robotic mitral valve plasty for mitral regurgitation after blunt chest trauma in Barlow's disease
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Norihiko Ishikawa Go Watanabe Tatsuya Tarui Takahumi Horikawa Ryuta Seguchi Ryuta Kiuchi Shigeyuki Tomita Hiroshi Ohtake Kenji Kawachi 《Asian journal of endoscopic surgery》2018,11(1):35-38
We successfully treated a case of mitral regurgitation due to chest trauma in Barlow's disease. A 71‐year‐old man was admitted with severe mitral regurgitation after blunt compression of the chest by a heavy object 5 months earlier. Preoperative examination revealed wide chordae tendineae rupture and myxomatous changes to the bileaflets. Neo‐chordae reconstruction of the anterior mitral leaflet using loop technique, triangular resection of the posterior mitral leaflet, and ring annuloplasty was performed via surgical robot. Robotic mitral valve plasty for severe mitral regurgitation due to chest trauma in Barlow's disease was achieved safely with good clinical and excellent cosmetic results. 相似文献
94.
Biomaterial characterization of off‐the‐shelf decellularized porcine pericardial tissue for use in prosthetic valvular applications
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Joshua A. Choe Soumen Jana Brandon J. Tefft Ryan S. Hennessy Jason Go David Morse Amir Lerman Melissa D. Young 《Journal of tissue engineering and regenerative medicine》2018,12(7):1608-1620
Fixed pericardial tissue is commonly used for commercially available xenograft valve implants, and has proven durability, but lacks the capability to remodel and grow. Decellularized porcine pericardial tissue has the promise to outperform fixed tissue and remodel, but the decellularization process has been shown to damage the collagen structure and reduce mechanical integrity of the tissue. Therefore, a comparison of uniaxial tensile properties was performed on decellularized, decellularized‐sterilized, fixed, and native porcine pericardial tissue versus native valve leaflet cusps. The results of non‐parametric analysis showed statistically significant differences (p < .05) between the stiffness of decellularized versus native pericardium and native cusps as well as fixed tissue, respectively; however, decellularized tissue showed large increases in elastic properties. Porosity testing of the tissues showed no statistical difference between decellularized and decell‐sterilized tissue compared with native cusps (p > .05). Scanning electron microscopy confirmed that valvular endothelial and interstitial cells colonized the decellularized pericardial surface when seeded and grown for 30 days in static culture. Collagen assays and transmission electron microscopy analysis showed limited reductions in collagen with processing; yet glycosaminoglycan assays showed great reductions in the processed pericardium relative to native cusps. Decellularized pericardium had comparatively low mechanical properties among the groups studied; yet the stiffness was comparatively similar to the native cusps and demonstrated a lack of cytotoxicity. Suture retention, accelerated wear, and hydrodynamic testing of prototype decellularized and decell‐sterilized valves showed positive functionality. Sterilized tissue could mimic valvular mechanical environment in vitro, therefore making it a viable potential candidate for off‐the‐shelf tissue‐engineered valvular applications. 相似文献
95.
Objectives
Since the Cabinet’s decision concerning the Basic Policies 2005, the Japanese government has implemented specific measures to suppress increases in national medical care expenditure. However, we believe that the economic significance of medical care should be quantified in terms of its economic impact on national medical care expenditure. No one has examined the economic impact of all medical institutions in Japan using data from a statement of profits and losses. We used an input–output analysis to quantitatively estimate economic impact of medical care and examined its estimation range with a probabilistic sensitivity analysis.Methods
To estimate the economic impact and economic impact multipliers of all medical institutions in Japan, an input–output analysis model was developed using an input–output table, statement of profits and losses, margin rates, employee income rates, consumption propensity and an equilibrium output model. Probabilistic sensitivity analysis was conducted using a Monte Carlo simulation.Results
Economic impact of medical care in all medical institutions was ¥72,107.4 billion ($661.5 billion). This impact yielded a 2.78-fold return of medical care expenditure with a 95 % confidence interval ranging from 2.74 to 2.90.Conclusion
Economic impact of medical care in Japan was two to three times the medical care expenditure (per unit). Production inducement of medical care is comparable to other industrial sectors that are highly influential toward the economy. The contribution to medical care should be evaluated more explicitly in national medical care expenditure policies. 相似文献96.
97.
Katsuyuki Miyabe Kenji Notohara Go Asano Akihisa Kato Naruomi Jinno Makoto Natsume Yasuki Hori Michihiro Yoshida Itaru Naitoh Ken Tsuboi Yoichi Matsuo Hirotaka Ohara Kazuki Hayashi Hiromi Kataoka 《Internal medicine (Tokyo, Japan)》2021,60(1):47
An 84-year-old man was admitted with epigastralgia. Computed tomography showed contrast-enhanced wall thickness in the cystic duct. An endoscopic examination revealed short irregular stricture in the cystic duct, and per-oral cholangioscopy revealed a reddish papillary tumor at the stricture site. Surgical resection revealed high-grade biliary intraepithelial neoplasia (BilIN) at the stricture site of the cystic duct. To our knowledge, this is the first case of a solitary high-grade BilIN epithelium in the cystic duct detected by per-oral cholangioscopy. 相似文献
98.
Ryohei Miyata Motohide Shimazu Shigeyuki Kawachi Minoru Tanabe Koichi Aiura Go Wakabayashi Masakazu Ueda Masayoshi Sakuma Masaki Kitajima 《Journal of hepato-biliary-pancreatic sciences》2005,12(3):272-276
A 54-year-old woman with giant liver cystadenocarcinoma underwent left trisegmentectomy with combined resection of the inferior vena cava (IVC) and the right hepatic vein. As a result, only the right inferior hepatic vein was preserved as a drainage vein. Because the perivertebral plexus and the azygos vein were both well developed, neither veno-venous bypass nor IVC reconstruction was performed. The developed collateral veins acted as the venous drainage pathway to maintain a stable systemic circulation. On the seventh postoperative day, portal vein flow dramatically decreased and the patient tended to liver failure. Prostaglandin E1 (PGE1) was administrated via the superior mesenteric artery. The portal flow then gradually increased and liver failure was avoided. Six months after the operation, she was re-admitted due to obstructive jaundice and presented with complete stenosis of the common bile duct (CBD). The jaundice persisted and liver dysfunction progressed. The patient died seven months after the operation. The confluence of the right inferior vein and the IVC could have been deformed, causing outflow blockade. The intrinsic shunt was not good enough to act as the drainage pathway, and IVC reconstruction may have been needed. 相似文献
99.
Lee WY Capra AM Jensvold NG Gurwitz JH Go AS;Epidemiology Practice Outcomes Cost of Heart Failure 《The American journal of cardiology》2004,94(9):1147-1152
Congestive heart failure (CHF) is the leading cause of hospitalization in the elderly, and these patients are at high risk for subsequent hospitalization. Whether gender affects the risk of rehospitalization in patients who have CHF is less well understood. We studied a random sample of 1,700 adults who had been hospitalized with CHF (from July 1, 1999 to June 30, 2000) and identified all readmissions through June 30, 2001. We used proportional hazards regression to evaluate whether gender affects the risk of all-cause and CHF-specific rehospitalization, after adjusting for differences in demographic characteristics, health-related behaviors, co-morbid conditions, left ventricular systolic function status, and use of CHF therapies. Among 1,591 adults who had confirmed CHF, 752 were women (47.3%). Women were older than men (73 vs 71 years, p <0.001) and more likely to have preserved systolic function (55.3% vs 40.9%, p <0.001), hypertension (83.1% vs 75.2%, p <0.001), and prior renal insufficiency (46.8% vs 34.6%, p <0.001). No significant differences existed between women and men with respect to crude rates of any readmission (144.7 vs 134.6 per 100 person-years, p = 0.36) or CHF-specific readmission (39.9 vs 37.4 per 100 person-years, p = 0.65). After adjusting for potential confounders, there was no significant difference between women and men with respect to risk of any readmission (adjusted hazard ratio 0.88, 95% confidence interval 0.76 to 1.02) or readmission for CHF (adjusted hazard ratio 0.89, 95% confidence interval 0.71 to 1.11). Among a contemporary, diverse population of patients who had CHF, rates of readmission overall and for CHF remained high, but gender was not independently associated with a differential risk of readmission. 相似文献
100.
Yuichi Minato Shiho Suzuki Tomoaki Hara Yutaka Kofuku Go Kasuya Yuichiro Fujiwara Shunsuke Igarashi Ei-ichiro Suzuki Osamu Nureki Motoyuki Hattori Takumi Ueda Ichio Shimada 《Proceedings of the National Academy of Sciences of the United States of America》2016,113(17):4741-4746
Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (<100 s−1), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.In chemical neurotransmission, various neurotransmitters bind to ligand-gated ion channels expressed in the plasma membrane of postsynaptic cells, such as the NMDA, AMPA, and P2X receptors, leading to changes in membrane potential and the concentration of intracellular ions. Each ligand for a ligand-gated ion channel has a distinct ability to evoke currents (1), and the ligands are classified according to the evoked current level: such as, full agonists, partial agonists, and antagonists. Partial agonists of ligand-gated ion channels reportedly offer clinical advantages over antagonists and full agonists in antidepressant and smoking-cessation treatment (2, 3).Two mechanisms have been proposed for the partial activation of the ligand-gated ion channels: the equilibrium between the open and closed conformations and the distinct conformation of the partial agonist-bound states from the closed and open conformations (4, 5). In the crystal structures of the extracellular region of the AMPA receptor, in which the distances between the two extracellular domains are changed upon agonist binding, the interdomain distances in the partial agonist-bound states correlated with the conductance level, suggesting that the AMPA receptor adopts specific intermediately permeable conformations (4, 6).The P2X receptors are a family of cation channels gated by extracellular ATP (1, 7–9) and are involved in many physiological and pathophysiological processes (10–12). Seven subtypes of the P2X receptors have been identified in mammals (13), and they share ∼40% sequence identity. The P2X4 receptor is involved in the pathogenesis of chronic neuropathic, inflammatory pain and the endothelial cell-mediated control of vascular tone (11, 14, 15). Compared with ATP, α,β-methylene ATP (α,β-meATP), in which the oxygen atom linking the α- and β-phosphorous atoms of ATP is replaced by a methylene group (Fig. S1A), reportedly induces a lower maximum current in cells expressing the mouse, rat, and human P2X4 receptors and other P2X receptors (16, 17).Open in a separate windowFig. S1.Characterization of the P2X4 receptor. (A) Chemical structures of ATP and α,β-meATP. (B and C) TEVC recordings of ATP- and α,β-meATP-evoked currents from rat P2X4 receptor expressed in Xenopus oocytes, respectively. In B, the currents were evoked twice by ATP (30 μM, 1 min, black bar). In C, the currents were firstly evoked by ATP (30 μM, 1 min, black bar) and subsequently by α,β-meATP (300 μM, 1 min, black bar). (D) TEVC recording of the ATP-evoked current (30 μM, 30 s, black bar) from the N-terminally EGFP-tagged ΔzfP2X4–A′ construct expressed in Xenopus oocytes. (E) Size exclusion chromatogram of purified EGFP-tagged ΔzfP2X4–A′ in rHDLs. Elution volumes corresponding to 17.0, 12.2, 10.4, and 7.1 nm Stokes diameters were determined by thyroglobulin, ferritin, catalase, and BSA, respectively. V0 and 1CV are void volume and single column volume, respectively. (F) SDS/PAGE analyses of purified ΔzfP2X4–A′ embedded in rHDLs. The samples were analyzed by 12% SDS/PAGE with Coomassie Brilliant Blue staining. (G) Measurement of [3H]ATP saturation binding to the purified ΔzfP2X4–A′ in rHDLs. (H and I) Estimation of the effects of deuteration based on the crystal structures of zfP2X4 (PDB ID code 4DW1) and the deuteration incorporation rates. The plots on the Left (without deuteration) and the Right (with deuteration) are the sums of the inverse sixth power of the distances between pseudoatoms centered on the methyl hydrogens of M108, M249, M268, or M325 and each hydrogen atom in the crystal structure of zfP2X4 (sums of the r−6) and the sums of the r−6 multiplied by [1 − (deuterium incorporation rates)] of each hydrogen atom, respectively. The graphs in H and I were calculated from the crystal structure in the apo state (PDB ID code 4DW0) and that in the ATP-bound state (PDB ID code 4DW1), respectively. Sums of the r−6 of each methionine methyl group and Hαβγ of the intraresidue methionine (green), Hαβγ of the interresidue methionine (light green), Hαβ of tyrosine (light violet), Hδεζη of tryptophan (orange), Hαβδεζ of phenylalanine (pink), Hαβγ of valine (blue), Hαβγδ of leucine (light blue), Hαβγδ of isoleucine (cyan), Hαβγ of threonine (light cyan), Hαβ of alanine (red), Hαβγδ of arginine (dark blue), Hα of glycine (dark green), and Hαβ of serine (magenta) residues, and the other hydrogens connected to carbon atoms (other unexchangeable hydrogens, light gray) are shown with colors. Hydrogen atoms connected to nitrogen, oxygen, or sulfur atoms were not considered in these calculations because these hydrogens should be exchanged with deuterium in D2O. The deuterium incorporation rates of the hydrogen atoms within each methionine residue (intraresidue) and the deuterium incorporation rates of other methionine residues (interresidue) were set to 98% and 85%, respectively, because the methionine residues would be derived from 85% of [α-, β-, γ-98% 2H-, methyl-13C]-methionine and 15% of nonlabeled methionine in the medium.The crystal structures of zebrafish P2X4 receptor (zfP2X4) (18, 19), together with mutational analyses (20–26), provided the structural basis for the channel opening of P2X receptors upon ATP binding. In the crystal structures, zfP2X4 forms a homotrimer (27, 28), in which the transmembrane region of each subunit is composed of two helices (19). In the crystal structure of zfP2X4 in the ATP-bound state, three ATP molecules are bound to the intersubunit nucleotide binding pockets. In addition, the region that connects the ATP-binding site and the transmembrane region, which is referred to as the “lower body” (Fig. 1 A and B), is expanded by ∼10 Å in the ATP-bound state, and a pore is formed in the transmembrane region, which is proposed to expand by the iris-like movement of the transmembrane helices (18). However, the mechanism underlying the partial activation of P2X receptors is unknown because the structures of the P2X receptors have not been examined in the partial agonist-bound states.Open in a separate windowFig. 1.NMR resonances from the endogenous methionine residues of zfP2X4 in rHDL. (A and B) Distribution of the methionine residues in the ΔzfP2X4–A′. One subunit from the crystal structure of zfP2X4 in the apo form (A) (PDB ID code 4DW0) and one from the ATP-bound form (B) (PDB ID code 4DW1) are shown in ribbons. The lower body and the right flipper are yellow. The A330 residues, the methionine residues, and the residues in which methionine mutations were introduced, L339 and L351, are depicted by green sticks. ATP is depicted by red sticks. Dummy atoms generated by Orientations of Proteins in Membranes (OPM), which represent membrane boundary planes, are gray. (C) Overlaid 1H-13C HMQC spectra of [2H-11AA, α, β-2H, methyl-13C-Met]ΔzfP2X4-A′, embedded in rHDLs, in the apo state (black) and the ATP-bound state (red). The regions with resonances from methionine residues are shown, and the assigned resonances are indicated. The centers of the resonances are indicated with dots. Cross-sections at lines through the centers of each resonance in the ATP-bound state and the cross-sections of the spectra using [α, β-2H, methyl-13C-Met]ΔzfP2X4-A′ are shown on the top of the overlaid spectra. The intensities of the cross-sections were normalized by the concentration of ΔzfP2X4-A′ and the conditions of the NMR measurements.The P2X4 receptor used in the previous crystallographic studies was solubilized by detergents, which are widely used for structural investigations of membrane proteins, but the P2X4 receptor is embedded in lipid bilayers under physiological conditions. It was recently reported that reconstituted high-density lipoproteins (rHDLs), which are also known as nanodiscs (29), can accommodate membrane proteins within a 10-nm-diameter disk-shaped lipid bilayer (30). The rHDLs reportedly provide a lipid environment with more native-like properties, compared with liposomes, in terms of the lateral pressure and curvature profiles because detergent micelles have strong curvature and different lateral pressure profiles from lipid membranes (31). Our NMR analyses of a G protein-coupled receptor (GPCR) and an ion channel in rHDL lipid bilayers revealed that the population and the exchange rates of the conformational equilibrium determine their signal transduction and ion transport activities (32–34) and that the population of the active conformation of the GPCR in rHDLs correlated better with the signaling levels than that in detergent micelles (32). Therefore, NMR investigations of membrane proteins in the lipid bilayer environments of rHDLs are necessary for accurate measurements of the exchange rates and the populations in conformational equilibrium.Here, we used NMR to observe the conformational equilibrium of the alanine and methionine residues of zfP2X4 bound to α,β-meATP in rHDLs. Based on the conformational equilibrium, we discuss the mechanism underlying the partial activation of P2X receptors. 相似文献