Vacuolar H+-ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases ...transport protons by a rotary catalytic mechanism analogous to that used by F1F0-ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmedin vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominant-negative mutants resulted in dramatic decreases in bafilomycin A1-sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase.
Osteoclasts form an acidic compartment at their attachment site in which bone demineralization and matrix degradation occur. Although both the cysteine proteinases and neutral collagenases ...participate in bone resorption, their roles have remained unclear. Here we show that interstitial collagenase has an essential role in initiating bone resorption, distinct from that of the cysteine proteinases. Treatment of osteoclasts with cysteine proteinase inhibitors did not affect the number of resorption lacunae (“pits”) formed on the surface of dentine slices, but it generated abnormal pits that were demineralized but filled with undegraded matrix. Treatment with metalloproteinase inhibitors did not alter the qualitative features of lacunae, but it greatly reduced the number of pits and surface area resorbed. Treatment of bone cells with an inhibitory anti-rat interstitial collagenase antiserum reduced bone resorption markedly. In the presence of collagenase inhibitors, resorption was restored by pretreatment of dentine slices with rat interstitial collagenase or by precoating the dentine slices with collagenase-derived gelatin peptides or heat-gelatinized collagen. Immunostaining revealed that interstitial collagenase is produced at high levels by stromal cells and osteoblasts adjacent to osteoclasts. These results indicate that interstitial collagenase can function as a “coupling factor,” allowing osteoblasts to initiate bone resorption by generating collagen fragments that activate osteoclasts.
The kidney adjusts net acid excretion to match production with exquisite precision, despite little or no change in the plasma bicarbonate concentration. The acid-sensing pathway that signals the ...kidney to increase acid secretion involves activation of the proto-oncogene c-Src. A new study in this issue shows that proline-rich tyrosine kinase 2 (Pyk2) is responsible for acid-induced activation of c-Src and is essential for acid sensing in renal epithelial cells. The findings implicate a broader role for Pyk2 in acid-base homeostasis in bone and other tissues beyond the kidney.
Background:
Carpal tunnel release (CTR) is a surgical treatment option for patients with carpal tunnel syndrome (CTS) symptoms that are unresponsive to conservative treatment. Most patients ...experience symptomatic relief after CTR regardless of the surgical technique. However, direct comparisons of the safety and effectiveness between CTR surgical techniques are limited. The purpose of this randomized controlled trial is to compare the safety and effectiveness of CTR with ultrasound guidance (CTR-US) versus mini-open CTR (mOCTR) in subjects with symptomatic CTS.
Design and methods:
TUTOR (Trial of Ultrasound guided CTR versus Traditional Open Release) is a randomized controlled trial in which 120 subjects at up to 12 sites in the United States will be randomized (2:1) to receive CTR-US or mOCTR. The primary endpoint of the study is the percentage of patients who return to normal daily activities within 3 days of the procedure. Secondary endpoints of the study are median time to return to normal daily activities, percentage of patients who return to work within 3 days of the procedure, median time to return to work, Boston Carpal Tunnel Questionnaire Symptom Severity Scale (BCTQ-SSS) change score at 3 months, BCTQ Functional Status Scale (BCTQ-FSS) change score at 3 months, Numeric Pain Scale change score at 3 months, EuroQoL-5 Dimension 5-Level (EQ-5D-5L) change score at 3 months, and the incidence of device- or procedure-related adverse events at 3 months. Patient follow-up in this trial will continue for 1 year.
Ethics and dissemination:
This study was approved by a central institutional review board and ongoing trial oversight will be provided by a data safety monitoring board (DSMB). The authors intend to report the results of this trial at medical conferences and peer-reviewed journals. The outcomes of TUTOR will have important clinical and economic implications for all stakeholders involved in treating patients with CTS.
Study registration:
ClinicalTrials.gov (https://clinicaltrials.gov): NCT05405218.
Level of evidence:
1
Vacuolar H(+)-ATPase (V-ATPase) binds microfilaments, and that interaction may be mediated by an actin binding domain in subunit B of the enzyme. To test for possible physiologic functions of the ...actin binding activity of V-ATPase, early responses of resorbing osteoclasts to inhibition of phosphatidylinositol 3-kinase activity by wortmannin and LY294002 were examined. Rapid co-localization between V-ATPase and F-actin was demonstrated by immunocytochemistry, and corresponding association between V-ATPase and F-actin in immunoprecipitations and pelleting assays was detected. This response was reversed as osteoclasts recovered resorptive activity after inhibitors were removed. By expressing and characterizing fusion proteins containing segments of the actin-binding amino-terminal regions of the B subunits of V-ATPase, we mapped the actin-binding site to a 44-amino acid domain. An 11-amino acid segment with a sequence similar to the actin-binding site of human profilin I was detected within this region. 13-Mers containing these profilin-like segments bound actin in fluorescent anisotropy studies and competed with profilin for binding to actin. Using site-directed mutagenesis, the 11-amino acid profilin-like actin-binding motifs (amino acids 49-59 of B1 and 55-65 of B2) were replaced with an 11-amino acid spacer with a sequence based on the homologous sequence from subunit B of Pyrococcus horikoshii, an organism that lacks an actin cytoskeleton. These substitutions eliminated the actin-binding activity of the B subunit fusion proteins. In summary, binding between V-ATPase and F-actin in osteoclasts occurs in response to blocking phosphatidylinositol 3-kinase activity. This response was fully reversible. The actin binding activities of the B subunits of V-ATPase required 11-amino acid actin-binding motifs that are similar in sequence to the actin-binding site of mammalian profilin I.
Context. Young planets are expected to cause cavities, spirals, and kinematic perturbations in protostellar disks that may be used to infer their presence. However, a clear detection of still-forming ...planets embedded within gas-rich disks is still rare. Aims. HD 169142 is a very young Herbig Ae-Be star surrounded by a pre-transitional disk, composed of at least three rings. While claims of sub-stellar objects around this star have been made previously, follow-up studies remain inconclusive. The complex structure of this disk is not yet well understood. Methods. We used the high contrast imager SPHERE at ESO Very large Telescope to obtain a sequence of high-resolution, high-contrast images of the immediate surroundings of this star over about three years in the wavelength range 0.95–2.25 μm. This enables a photometric and astrometric analysis of the structures in the disk. Results. While we were unable to definitively confirm the previous claims of a massive sub-stellar object at 0.1–0.15 arcsec from the star, we found both spirals and blobs within the disk. The spiral pattern may be explained as due to the presence of a primary, a secondary, and a tertiary arm excited by a planet of a few Jupiter masses lying along the primary arm, likely in the cavities between the rings. The blobs orbit the star consistently with Keplerian motion, allowing a dynamical determination of the mass of the star. While most of these blobs are located within the rings, we found that one of them lies in the cavity between the rings, along the primary arm of the spiral design. Conclusions. This blob might be due to a planet that might also be responsible for the spiral pattern observed within the rings and for the cavity between the two rings. The planet itself is not detected at short wavelengths, where we only see a dust cloud illuminated by stellar light, but the planetary photosphere might be responsible for the emission observed in the K1 and K2 bands. The mass ofthis putative planet may be constrained using photometric and dynamical arguments. While uncertainties are large, the mass should be between 1 and 4 Jupiter masses. The brightest blobs are found at the 1:2 resonance with this putative planet.
Regulation of AE1 anion exchanger and H+-ATPase in rat cortex by acute metabolic acidosis and alkalosis. The cortical collecting duct (CCD) mediates net secretion or reabsorption of protons according ...to systemic acid/base status. Using indirect immunofluorescence, we examined the localization and abundance of the vacuolar H+-ATPase and the AE1 anion exchanger in intercalated cells (IC) of rat kidney connecting segment (CNT) and CCD during acute (6 hr) metabolic (NH4Cl) acidosis and respiratory (NaHCO3) alkalosis. AE1 immunostaining intensity quantified by confocal microscopy was elevated in metabolic acidosis and substantially reduced in metabolic alkalosis. AE1 immunostaining was restricted to Type A IC in all conditions, and the fraction of AE1+ IC was unchanged in CNT and CCD. Metabolic acidosis was accompanied by redistribution of H+-ATPase immunostaining towards the apical surface of IC, and metabolic alkalosis was accompanied by H+-ATPase redistribution towards the basal surface of IC. Therefore, acute metabolic acidosis produced changes consistent with increased activity of Type A IC and decreased activity of Type B IC, whereas acute metabolic alkalosis produced changes corresponding to increased activity of Type B IC and decreased activity of Type A IC. These data demonstrate that acute systemic acidosis and alkalosis modulate the cellular distribution of two key transporters involved in proton secretion in the distal nephron.
Comparative studies of carpal tunnel release with ultrasound guidance (CTR-US) vs. mini-open CTR (mOCTR) are limited, prompting development of this randomized trial to compare efficacy and safety of ...these techniques.
Patients were randomized (2:1) to CTR-US or mOCTR, treated by experienced hand surgeons (median previous cases: 12 CTR-US; 1000 mOCTR), and followed for 3 months.
Among 149 randomized patients, 122 received CTR-US (n = 94) or mOCTR (n = 28). Mean incision length was 6 ± 2 mm in the wrist (CTR-US) vs. 22 ± 7 mm in the palm (mOCTR) (p < 0.001). Median time to return to daily activities (2 vs. 2 days; p = 0.81) and work (3 vs. 4 days; p = 0.61) were similar. Both groups reported statistically significant and clinically important improvements in Boston Carpal Tunnel Questionnaire Symptom Severity and Functional Status Scales, Numeric Pain Scale, and EuroQoL-5 Dimension 5-Level, with no statistical differences between groups. Freedom from wound sensitivity and pain favored CTR-US (61.1% vs. 17.9%; p < 0.001). Adverse event rates were low in each group (2.1% vs. 3.6%; p = 0.55).
The efficacy and safety of CTR-US were comparable to mOCTR despite less previous surgical experience with CTR-US. The choice of CTR technique should be determined by shared decision-making between patient and physician.
www.clinicaltrials.gov
identifier is NCT05405218.
The SpHere INfrared Exoplanet (SHINE) project is a 500-star survey performed with SPHERE on the Very Large Telescope for the purpose of directly detecting new substellar companions and understanding ...their formation and early evolution. Here we present an initial statistical analysis for a subsample of 150 stars spanning spectral types from B to M that are representative of the full SHINE sample. Our goal is to constrain the frequency of substellar companions with masses between 1 and 75
M
Jup
and semimajor axes between 5 and 300 au. For this purpose, we adopt detection limits as a function of angular separation from the survey data for all stars converted into mass and projected orbital separation using the BEX-COND-hot evolutionary tracks and known distance to each system. Based on the results obtained for each star and on the 13 detections in the sample, we use a Markov chain Monte Carlo tool to compare our observations to two different types of models. The first is a parametric model based on observational constraints, and the second type are numerical models that combine advanced core accretion and gravitational instability planet population synthesis. Using the parametric model, we show that the frequencies of systems with at least one substellar companion are 23.0
−9.7
+13.5
, 5.8
−2.8
+4.7
, and 12.6
−7.1
+12.9
% for BA, FGK, and M stars, respectively. We also demonstrate that a planet-like formation pathway probably dominates the mass range from 1–75
M
Jup
for companions around BA stars, while for M dwarfs, brown dwarf binaries dominate detections. In contrast, a combination of binary star-like and planet-like formation is required to best fit the observations for FGK stars. Using our population model and restricting our sample to FGK stars, we derive a frequency of 5.7
−2.8
+3.8
%, consistent with predictions from the parametric model. More generally, the frequency values that we derive are in excellent agreement with values obtained in previous studies.
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