Structure of the rotor ring modified with N,N'-dicyclohexylcarbodiimide of the Na+-transporting vacuolar ATPase.

The prokaryotic V-ATPase of Enterococcus hirae, closely related to the eukaryotic enzymes, provides a unique opportunity to study the ion-translocation mechanism because it transports Na(+), which can be detected by radioisotope (22Na(+)) experiments and X-ray crystallography. In this study, we demonstrated that the binding affinity of the rotor ring (K ring) for 22Na(+) decreased approximately 30-fold by reaction with N,N(')-dicyclohexylcarbodiimide (DCCD), and determined the crystal structures of Na(+)-bound and Na(+)-unbound K rings modified with DCCD at 2.4- and 3.1-Å resolutions, respectively. Overall these structures were similar, indicating that there is no global conformational change associated with release of Na(+) from the DCCD-K ring. A conserved glutamate residue (E139) within all 10 ion-binding pockets of the K ring was neutralized by modification with DCCD, and formed an "open" conformation by losing hydrogen bonds with the Y68 and T64 side chains, resulting in low affinity for Na(+). This open conformation is likely to be comparable to that of neutralized E139 forming a salt bridge with the conserved arginine of the stator during the ion-translocation process. Based on these findings, we proposed the ion-translocation model that the binding affinity for Na(+) decreases due to the neutralization of E139, thus releasing bound Na(+), and that the structures of Na(+)-bound and Na(+)-unbound DCCD-K rings are corresponding to intermediate states before and after release of Na(+) during rotational catalysis of V-ATPase, respectively.

Difference in Physiological Components of VO2 Max During Incremental and Constant Exercise Protocols for the Cardiopulmonary Exercise Test.

[Purpose] VO2 is expressed as the product of cardiac output and O2 extraction by the Fick equation. During the incremental exercise test and constant high-intensity exercise test, VO2 results in the attainment of maximal O2 uptake at exhaustion. However, the differences in the physiological components, cardiac output and muscle O2 extraction, have not been fully elucidated. We tested the hypothesis that constant exercise would result in higher O2 extraction than incremental exercise at exhaustion. [Subjects] Twenty-five subjects performed incremental exercise and constant exercise at 80% of their peak work rate. [Methods] Ventilatory, cardiovascular, and muscle oxygenation responses were measured using a gas analyzer, Finapres, and near-infrared spectroscopy, respectively. [Results] VO2 was not significantly different between the incremental exercise and constant exercise. However, cardiac output and muscle O2 saturation were significantly lower for the constant exercise than the incremental exercise at the end of exercise. [Conclusion] These findings indicate that if both tests produce a similar VO2 value, the VO2 in incremental exercise would have a higher ratio of cardiac output than constant exercise, and VO2 in constant exercise would have a higher ratio of O2 extraction than incremental exercise at the end of exercise.

Cardiac and renal protective effects of 2,5-dimethylcelecoxib in angiotensin II and high-salt-induced hypertension model mice.

BACKGROUND: We reported that 2,5-dimethylcelecoxib (DM-celecoxib), a celecoxib derivative that is unable to inhibit cyclooxygenase-2, prevented cardiac remodeling induced by sarcomeric gene mutation, left ventricular pressure overload, or ß-adrenergic receptor stimulation. This effect seemed to be mediated by the inhibition of the canonical Wnt/ß-catenin signaling pathway, which has been suggested to play a key role in the development of chronic kidney disease and chronic heart failure. METHOD: We investigated the effect of DM-celecoxib on cardiac remodeling and kidney injury in hypertension model mice induced by angiotensin II infusion in the absence or presence of high-salt load. RESULTS: DM-celecoxib prevented cardiac remodeling and markedly reduced urinary albumin excretion without altering blood pressure in those mice. Moreover, DM-celecoxib prevented podocyte injury, glomerulosclerosis, and interstitial fibrosis in the kidney of mice loaded with angiotensin II and high-salt load. DM-celecoxib reduced the phosphorylation level of Akt and activated glycogen synthase kinase-3, which led to the suppression of the Wnt/ß-catenin signal in the heart and kidney. DM-celecoxib also reduced the expression level of snail, a key transcription factor for the epithelial-mesenchymal transition and of which gene is a target of the Wnt/ß-catenin signal. CONCLUSION: Results of the current study suggested that DM-celecoxib could be beneficial for patients with hypertensive heart and kidney diseases.

Solvation Structures of Tetraethylammonium Bromide and Tetrafluoroborate in Aqueous Binary Solvents with Ethanol, Trifluoroethanol, and Acetonitrile.

The solvation structures of tetraethylammonium bromide and tetrafluoroborate (TEABr and TEABF4) in aqueous binary solvents with ethanol (EtOH), 2,2,2-trifluoroethanol (TFE), and acetonitrile (AN) have been clarified by molecular dynamics (MD) simulations. In addition, 1H and 13C NMR chemical shifts of the H and C atoms within TEA+ in the binary solvents have been measured as a function of the mole fraction of the organic solvent, xOS. The variations of the chemical shifts with an increase in xOS were interpreted according to the solvation structures of TEA+, Br-, and BF4- obtained from the MD simulations. It has been found that TEABF4 at 130 mmol dm-3 cannot be dissolved into the EtOH and TFE solvents above xOS ≈ 0.7 and 0.6, respectively, while TEABr can be done in both solvents. Interestingly, TEABr and TEABF4 at the concentration can be dissolved in the AN solvents over the entire xOS range. The solvation of TEA+, Br-, and BF4- in each solvent has been discussed in terms of the electrostatic force, the weak hydrogen bond of C-H···F-C, and the dipole-dipole interaction.

A Perforating Artery Compressing the Nerve Rootlet and Causing Glossopharyngeal Neuralgia.

BACKGROUND: A surgical procedure for glossopharyngeal neuralgia (GPN) was selected from microvascular decompression, glossopharyngeal and upper vagal rhizotomy, or a combination of these procedures based on the presence of arteries compressing the glossopharyngeal and vagal rootlets. The offending artery is usually a main trunk or branch of the cerebellar arteries. A perforating artery is a known but uncommon variation of the offending artery that causes GPN. The appropriate procedure for such cases is unknown. OBJECTIVE: To analyze the clinical significance of the perforating artery in GPN, we describe 2 patients with a perforating artery compressing the rootlet, and its mobilization relieved neuralgia. We examined the validity of decompressing a perforating artery as an alternative to rhizotomy in such cases. METHODS: We independently reviewed 12 GPN patients treated with microvascular decompression. The patients' pain severity, medication doses, preoperative imaging studies, intraoperative findings, and outcomes were examined. RESULTS: Eleven patients had neurovascular compression of the glossopharyngeal nerve. In 2 of the patients, a perforating artery compressed the rootlet, thereby generating an indentation and creating a discoloration of the rootlet. Mobilizing the perforating artery with no additional rhizotomy provided complete pain relief with no significant complications and allowed the discontinuation of medications. CONCLUSION: Even a small perforating artery can cause GPN when it compresses the rootlet. In such cases, mobilization of the perforating artery with no additional rhizotomy is an effective surgical option.

Regenerative treatment for soft tissue defects of the external auditory meatus.

OBJECTIVE: To establish a regenerative treatment for soft tissue defects of the external auditory meatus (EAM) without conventional surgical therapy. STUDY DESIGN: Controlled clinical pilot study. SETTING: General hospitals. PATIENTS: Sixty-five patients with new or old EAM defects without active inflammation were selected. Ages ranged from 12 to 87 years (average age of 58 yr). INTERVENTION: Therapeutic nonsurgical treatment of EAM defects. Gelatin sponge, basic fibroblast growth factor (b-FGF), fibrin glue, and water proof transparent dressing were used in the repair procedure. Patients were divided into 2 groups: treatment with (n = 54) and without (n = 11) b-FGF. After mechanically disrupting the edge of the EAM defect, gelatin sponge immersed in b-FGF was placed over the defect and covered with fibrin glue. In cases of extensive EAM defects, the EAM was filled with gelatin sponge/b-FGF, and the auricle was wrapped in water proof dressing. Two or 3 weeks postprocedure, crust over the defect was removed. If complete defect closure was not achieved after 1 treatment course, the treatment was repeated. MAIN OUTCOME MEASURE: Evaluation of complete closure of EAM defects 3 months posttreatment. RESULTS: Complete closure of the EAM defect was achieved within 3 treatment courses in 92.6% (50/54) and 18.2% (2/11) of the patients with or without b-FGF, respectively. No inflammation/infection or severe sequelae were observed. CONCLUSION: This study demonstrated the effectiveness of combining gelatin sponge, b-FGF, and fibrin glue for EAM defect regeneration. This innovative regenerative therapy is an easy, simple, cost-effective and minimally invasive method for treating EAM defects.

Interaction and stoichiometry of the peripheral stalk subunits NtpE and NtpF and the N-terminal hydrophilic domain of NtpI of Enterococcus hirae V-ATPase.

The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpI(Nterm)) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpI(Nterm) directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI(Nterm) bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI(Nterm) and NtpF. Purified NtpE-F-I(Nterm) complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated "L" shape.