The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl-channel regulated by protein kinase A. The most common mutation In cystic fibrosis (CF), deletion of Phe-S08 (AF508-CFTR), reduces Cl1 secretion, but the fatal consequences of CF have been difficult to rationalie solely in terms ofthis defect. The aim ofthis study was to determine the role of CFTR in HCO_ transport across cell membranes. HCO perbili was asse from measurements of intracellular pH [pHi; from spectrofluorimetry of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-S-(and -6)carboxyfluorescein] and of channel activity (patch damp; cell attached and isolated, inside-out patches) on NIH 3T3 fibroblasts and C127 mammary epithelial cells transfected with wild-type CFTR (WT-CFTR) or AF5S08-CFTR, and also on mock-transfected cells. When WT-CFTR-transfected cells were acidified (pulsed with NH4CI) and incubated in Na+-free (N-methyl-n-glucmine substitution) solutions (to block Na+-dependent pHI, regulatory mechanisms), pH, remained acidic (pH 6.5) until the cells were treated with 20 IAM forskolin (increases cellular [cAMPI); pHi then increased toward (but not completely to) control level (pH1 7.2) at a rate of 0.055 pH unit/min. Forskolin had no effect on rate of pHi recovery in AF508 and mock-transfected cells. This Na+-independent, forskolin-dependent pHi recovery was not observed in HCO /CO2-free medium. Forskolin-treated WT-CFTRtransfected (but not AF508-CFTR or mock-transfected) cells in Cl0-containing, HCO--free solutions showed Cl-channels with a linear I/V relationship and a conductance of 10.4 ± 0.5 pS in symmetrical 150 mM Cl-. When channeis were incubated with different [Cl-I and [HCOi-on the inside and outside, the Cl-/HCO; permeability ratio (determined from reversal potentials of i/V curves) was 3.8 ± 1.0 (mean ± SEM; n = 9); the ratio of conductances was 3.9 ± 0.5 (at 150 mM Cl-and 127 mM HCO5. We conclude that in acidified cells the WT-CFTR functions as a base loader by allowing a cAMPdependent influx of HCO-through channeis that conduct HCOj about one-quarter as efflciently as it conducts Cl-.Under physiological conditions, the electrochemical gradients for both Cl-and HCO5 are directed outward, so CFTR likely contributes to the epithellal secretion of both ions. HCO-3 secretion may be important for controlling pH of the luminal, but probably not the cytoplasmic, fluid in CFTR-containing epithelia. In CF, a decreased secretion of HCO3 may lead to decreased pH of the luminal fluid.The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl-channel regulated by cAMPdependent protein kinase A (1-3). The most common mutation in CF is deletion of Phe-508 of the CFTR (AF508-CFTR), which reduces the ability ofcells to secrete Cl-(4). However, the fatal consequences in CF have been difficult to rationalize solely as a defect in Cl-secretion (5, 6). If the CFTR were also conductive to HCO-, then movements of HCO-might affect the pH of the luminal solution of some epithelia. For example, i...

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme’s active site yields insights into the mechanism of action of this important class of enzymes.

We observed a marked decrease in quadriceps volume within the first week of intensive care for septic shock. This loss of muscle mass was unaffected by transcutaneous electrical muscle stimulation applied for 60 mins per day for 7 days.

No abstract

An extremely low-field signal (at approximately 18 p.p.m.) in the 1 H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wildtype RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theoretical calculations of chemical shift values were used in the interpretation of the experimental 1 H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 Å resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 Å ) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor-acceptor distances of different types of hydrogen bonds. The short hydrogen-bond interactions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pK a values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homologyreduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins.

Physical function is substantially reduced in survivors of septic shock 1 year after discharge.

SUMMARY1. The rate of acetylcholine (ACh)-induced fluid secretion was measured from the main excretory duct of rabbit lacrimal glands perfused in vivo with Krebs Henseleit bicarbonate solutions.2. Perfusion with ouabain (10-5 M) decreased the rate of lacrimal gland fluid secretion to 23 % of the control value.3. Perfusion with furosemide (10-4 and 1O-3 M), which has been shown to inhibit the coupled transport of Na+ and Cl-, reversibly decreased the rate of secretion to 43 and 33 % of the control value respectively. 6. It is concluded that ACh-induced secretion of electrolytes and water is dependent upon (Na+ + K+)-activated ATPase. In addition, coupled transport of Na+ and Cl-appears to be involved in secretion.7. Basolateral location of the (Na+ + K+)-activated ATPase implies that it plays an indirect role in electrolyte and water secretion. A possible role may be to energize a secondary active transport of Cl-that is mediated by a NaCl cotransport system.

Stimulation-induced changes in Cl- content and O2 consumption of collagenase-isolated rat parotid acini were measured. In less than 10 s, carbachol caused a net Cl- efflux, corresponding to approximately 50% of the Cl- content, and increased the O2 uptake by 100%. The increase was inhibitable by ouabain and was dependent on the presence of extracellular Ca2+. Furosemide reduced the unstimulated 36Cl- uptake and prevented the reuptake of Cl- after carbachol-induced release. This suggests that a cotransport system is operating in both the unstimulated and stimulated states. Furthermore, furosemide inhibited the stimulated ouabain-sensitive O2 uptake. Raising intracellular Ca2+ by the calcium ionophore A23187 evoked the same pattern of Cl- loss and O2 uptake as carbachol. Our results are compatible with the following hypothesis. Carbachol raises intracellular Ca2+, causing an increased Cl- permeability of the luminal membrane, resulting in a net Cl- efflux. A subsequently enhanced influx of Cl- and Na+ via a furosemide-sensitive cotransport system increases intracellular Na+. This stimulates the Na+-K+-ATPase and thereby the O2 consumption.