Biomedical subjects
Publications and source records attributed to W Hu.
An effective methodology is reported for stereospecific epoxidation and aziridination via carbonyl ylide intermediates using rhodium(II) acetate catalyzed reactions of phenyl- and styryldiazoacetates with aldehydes, ketones, or imines.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)-activated chloride channel that is localized to the plasma membrane and endosomal compartment. Endosomal targeting of CFTR is attributed to the Tyr(1424)-based internalization signal, identified in the C-terminal tail of the channel. Mutation of the Tyr(1424) residue could partly inhibit the endocytosis of CFTR and its association with the adapter protein AP-2. To reveal additional endosomal targeting signals, site-directed mutagenesis of both a chimaera, composed of a truncated form of interleukin 2 receptor alpha chain (TacT) and the C-terminal tail of CFTR (Ct), and the full-length CFTR was performed. Morphological and functional assays revealed the presence of multiple internalization motifs at the C-terminus, consisting of a phenylalanine-based motif (Phe(1413)) and a bipartite endocytic signal, comprising a tyrosine (Tyr(1424)) and a di-Leu-based (Leu(1430)-Leu) motif. Whereas the replacement of any one of the three internalization motifs with alanine prevented the endocytosis of the TacT-Ct chimaera, mutagenesis of Phe(1413)-Leu impaired the biosynthetic processing of CFTR, indicating that Phe(1413) is indispensable for the native structure of CFTR. In contrast, replacement of Leu(1430)-Leu- and Tyr(1424)-based signals with alanine increased the cell-surface density of both the chimaeras and CFTR in an additive manner. These results suggest that the internalization of CFTR is regulated by multiple endocytic sorting signals.
BACKGROUND: Unilateral audiovestibular symptoms are commonly seen in clinical practice and are rarely caused by retrocochlear pathology. However, clinicians are often required to rule out potentially serious causes of these unilateral symptoms. Gadolinium enhanced magnetic resonance imaging (GdMRI) is the most accurate test for detecting small cerebellopontine angle lesions and also screens the adjacent CNS structures. Its main disadvantage is the cost of the procedure. METHODS: We studied 100 consecutive patients with both GdMRI and a newer MRI screening study utilizing unenhanced T2-weighted fast spin echo (fse) MRI. Acquired images were randomly assessed by a panel of three neuro-radiologists. RESULTS: We found that the screening (fse) MRI was as sensitive and specific when detecting cerebellopontine angle tumors. CONCLUSIONS: We conclude that T2-weighted fse MRI is a safe and cost-effective alternative to GdMRI and offers better diagnostic utility when compared to auditory brain stem response (ABR) and CT scans.
We have used NMR spectroscopy to determine the solution structure of a complex between an oligonucleotide derived from stem IIB of the Rev responsive element (RRE-IIB) of HIV-1 mRNA and an in vivo selected, high affinity binding Arg-rich peptide. The peptide binds in a partially alpha-helical conformation into a pocket within the RNA deep groove. Comparison with the structure of a complex between an alpha-helical Rev peptide and RRE-IIB reveals that the sequence of the bound peptide determines the local conformation of the RRE peptide binding site. A conformational switch of an unpaired uridine base was revealed; this points out into the solvent in the Rev peptide complex, but it is stabilized inside the RNA deep groove by stacking with an Arg side chain in the selected peptide complex. The conformational switch has been visualized by NMR chemical shift mapping of the uridine H5/H6 atoms during a competition experiment in which Rev peptide was displaced from RRE-IIB by the higher affinity binding selected peptide.
Hypersensitive reaction and pathogenicity (hrp) genes are required for Pseudomonas syringae pv. tomato (Pst) DC3000 to cause disease in susceptible tomato and Arabidopsis thaliana plants and to elicit the hypersensitive response in resistant plants. The hrp genes encode a type III protein secretion system known as the Hrp system, which in Pst DC3000 secretes HrpA, HrpZ, HrpW, and AvrPto and assembles a surface appendage, named the Hrp pilus, in hrp-gene-inducing minimal medium. HrpA has been suggested to be the Hrp pilus structural protein on the basis of copurification and mutational analyses. In this study, we show that an antibody against HrpA efficiently labeled Hrp pili, whereas antibodies against HrpW and HrpZ did not. Immunogold labeling of bacteria-infected Arabidopsis thaliana leaf tissue with an Hrp pilus antibody revealed a characteristic lineup of gold particles around bacteria and/or at the bacterium-plant contact site. These results confirm that HrpA is the major structural protein of the Hrp pilus and provide evidence that Hrp pili are assembled in vitro and in planta.
The effect of food on the pharmacokinetics of 15 mg oxybutynin XL was evaluated in a single-dose, randomized, crossover, open-label study in healthy volunteers. A validated, stereospecific, high-performance liquid chromatography assay was used to simultaneously determine the plasma concentrations of R- and S-oxybutynin and active metabolite R- and S-desethyloxybutynin. The mean AUC and Cmax values for each of the four analytes in the fed treatment were within +/- 20% of the fasting treatment values. The 90% confidence intervals for the treatment ratios (fed/fasted) for log-transformed Cmax and AUCinf values for the drug isomers and AUCinf values for the metabolite isomers were all within the 80% to 125% range. Only the ranges for the Cmax values for R- and S-desethyloxybutynin were slightly wider but were well within the 70% to 143% criteria recommended for Cmax when comparing effect of food. Lack of effect of food on oxybutynin XL is consistent with the previous observation that the osmotically controlled formulations are nearly insensitive to the gastrointestinal environment, including food. Oxybutynin XL was well tolerated, and the safety results were comparable whether administered alone or with food. In conclusion, oxybutynin XL administration does not require any caution to be exercised regarding food.
Catalytic reactions of diazoacetates tethered through zero, one, two, and three ethylene glycol units to an allyl group have been investigated for chemoselectivity, diastereoselectivity, and enantioselectivity. Results from cyclopropanation, carbon-hydrogen insertion, and oxonium ylide generation are compared from reactions of achiral and chiral catalysts of copper(I) and dirhodium(II) carboxylates and carboxamidates. Relative to results from intermolecular reactions of ethyl diazoacetate with allyl ethyl ether, intermolecular reactions show a diversity of selectivities including preference for the opposite configurational arrangement from the one preferred in corresponding intermolecular cyclopropanation reactions. Enantioselectivities for cyclopropanation are dependent on the catalyst ligands in a manner that reflects divergent trajectories of the carbon-carbon double bond to the reacting carbene center. Enantioselectivity increases as a function of ring size with chiral copper catalysts, but the reverse occurs with chiral dirhodium(II) carboxamidates. Mechanistic implications, including those related to the conformation of the reacting metal carbene, offer a new dimension to understanding of enantioselectivity in catalytic asymmetric cyclopropanation reactions.
Deletion of phenylalanine at position 508 (DeltaF508) is the most common cystic fibrosis (CF)-associated mutation in the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The consensus notion is that DeltaF508 imposes a temperature-sensitive folding defect and targets newly synthesized CFTR for degradation at endoplasmic reticulum (ER). A limited amount of CFTR activity, however, appears at the cell surface in the epithelia of homozygous DeltaF508 CFTR mice and patients, suggesting that the ER retention is not absolute in native tissues. To further elucidate the reasons behind the inability of DeltaF508 CFTR to accumulate at the plasma membrane, its stability was determined subsequent to escape from the ER, induced by reduced temperature and glycerol. Biochemical and functional measurements show that rescued DeltaF508 CFTR has a temperature-sensitive stability defect in post-ER compartments, including the cell surface. The more than 4-20-fold accelerated degradation rate between 37 and 40 degrees C is, most likely, due to decreased conformational stability of the rescued DeltaF508 CFTR, demonstrated by in situ protease susceptibility and SDS-resistant thermoaggregation assays. We propose that the decreased stability of the spontaneously or pharmacologically rescued mutant may contribute to its inability to accumulate at the cell surface. Thus, therapeutic efforts to correct the folding defect should be combined with stabilization of the native DeltaF508 CFTR.
The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor phosphorylation, indicating that PKC-mediated phosphorylation occurs at Ser-344. PKC-mediated Ser-344 phosphorylation was also induced by activation of G(q)-coupled alpha(1A)-adrenergic receptor or increase in intracellular Ca(2+) concentration. Activation of PKC by PMA, alpha(1A)-adrenergic receptor agonist, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism. Further study demonstrated that agonist-dependent G protein-coupled receptor kinase (GRK) phosphorylation sites in DOR are not targets of PKC. Agonist-dependent, GRK-mediated receptor phosphorylation and agonist-independent, PKC-mediated DOR phosphorylation were additive, but agonist-induced receptor phosphorylation could inhibit PKC-catalyzed heterologous DOR phosphorylation and subsequent internalization. These data demonstrate that the responsiveness of opioid receptor is regulated by both PKC and GRK through agonist-dependent and agonist-independent mechanisms and PKC-mediated receptor phosphorylation is an important molecular mechanism of heterologous regulation of opioid receptor functions.
Explore the source record for details and available documents.
Cold dark matter (CDM) models predict small-scale structure in excess of observations of the cores and abundance of dwarf galaxies. These problems might be solved, and the virtues of CDM models retained, even without postulating ad hoc dark matter particle or field interactions, if the dark matter is composed of ultralight scalar particles (m approximately 10(-22) eV), initially in a (cold) Bose-Einstein condensate, similar to axion dark matter models. The wave properties of the dark matter stabilize gravitational collapse, providing halo cores and sharply suppressing small-scale linear power.
A simple, selective, and sensitive method for the simultaneous determination of anions (sulfate, nitrate, and chloride) and cations (sodium, ammonium, potassium, magnesium, and calcium) in acid rain waters was developed using ion-exclusion/ cation-exchange chromatography with conductimetric detection. A weakly acidic cation-exchange resin column (Tosho TSKgel OA-PAK-A) and a sulfosalicylic acid-methanol-water eluent was used. With a mobile phase comprising 1.25 mM sulfosalicylic acid in methanol-water (7.5:92.5) at 1.2 ml/min, simultaneous separation and detection of the above anions and cations was achieved in about 30 min. Linear calibration plots of peak area versus concentration were obtained over the concentration ranges 0-1.0 mM for anions (R=0.9991) and 0-0.5 mM for cations (R=0.9994). Detection limits calculated at S/N=3 ranged from 4.2 to 14.8 ppb for the anions and from 2.4 to 12.1 ppb for the cations. The reproducibility of retention times was 0.14-0.15% relative standard deviation (RSD) for anions and 0.18-0.31% for cations, and reproducibility of chromatographic peak areas was 1.22-1.75% RSD for anions and 1.81-2.10% for cations. The method was applied successfully to the simultaneous determination of anions and cations in aerosols transported from mainland China to central Japan, as determined by a meteorological satellite data analyzer.
Tetraborate is investigated as the eluent ion for suppressed electrostatic ion chromatography (EIC) using a zwitterionic stationary phase. Good separation of a range of inorganic anions (SO4(2-), Cl-, NO3-, Br-, NO3-, ClO3-, and I-) was obtained, with detection limits for highly conducting ions (SO4(2-), Cl-, NO2-, Br- , and NO3-) being less than 8 x 10(-8) M, and for weakly conducting anions (ClO3- and I-) being 2.7 x 10(-7) and 5.8 x 10(-7) M, respectively. Calibration curves were linear up to 1.8 mM of each analyte. Retention times were found to increase with increasing eluent concentration and a linear relationship was observed between log k' and log[Na2B4O7] for all analytes. This behaviour is attributed to the progressive formation of a binary electrical double layer at the surface of the zwitterionic stationary phase. Retention times of analytes could be manipulated by varying the concentration of the eluent. This new suppressed-EIC system was applied to the determination of inorganic anions (SO4(-2) , CI-, NO3-, NO2-, and Br-) in snow and rainwater samples.
Changes in sodium, potassium, and water content in brain tissue are important in the progression of pathology that follows ischemic stroke. Determining these parameters regionally in rodent models of experimental ischemia has been limited because typical tissue weights of more than 35 mg are too large. Identifying ischemic tissue to direct tissue sampling towards ischemic cortex is also represents a difficult generally unresolved area. We suggest that larger differences between normal and ischemic cortex of sodium, potassium, and water content than previously observed can be obtained from directed sampling of 2-mg brain tissue in a model of focal cerebral ischemia. In five rats, the middle cerebral artery and both common carotid arteries were occluded for 4.9+/-0.13 h (mean+/-SEM). Punch-sampling of 1-mm diameter tissue cores for water content (H(2)O%) by the wet-dry method, and [Na(+)] and [K(+)] by flame photometry, was guided by the observation of a subtle change in the surface reflectivity of ischemic cortex of quickly dried, 20-microm frozen brain sections, that was confirmed by MAP2 immunohistochemistry. The ratio of the lesion areas as determined by the reflective change and MAP2 immunoreactivity was 0.96+/-0.03 (n=5). In ischemic cortex H(2)O% was 79.9%+/-0.8%, [Na(+)] was 550+/-25 mEq/kg dry-weight, and [K(+)] 94.2+/-19.2 mEq/kg dry-weight (n=5), all significantly different from the values in border zone cortex, and in cortex contralateral to ischemic cortex and border zone (for all samples n=60, mean wet weight 2.037+/-0.046 mg). Differences between ischemic and normal cortex were 5.4+/-1.1%, 317+/-21 mEq/kg dry-weight, -304+/-27 mEq/kg dry-weight (n=5) for H(2)O%, [Na(+)], and [K(+)]. These differences between ischemic and normal cortex are 1.4-2.5, 1-3.11, and 1.4-3.5 times greater, respectively, than previous results obtained using samples weighing 35 mg or more. These results extend the association of sodium and potassium with ischemic brain edema in the rodent model, and show that these classical measurements can keep pace with the regionality of histochemical and morphological methods.
[reaction: see text] Effective synthetic uses of bisdiazocarbonyl compounds for the selective construction of diverse macrocycles, including the synthesis of patulolides A and B, by catalytic "carbene dimer" formation are reported. Control of stereochemistry and efficient methods for product isomerization or kinetic isomer differentiation have been achieved.
Weak gravitational lensing observations probe the spectrum and evolution of density fluctuations and the cosmological parameters that govern them, but they are currently limited to small fields and subject to selection biases. We show how the expected signal from large-scale structure arises from the contributions from and correlations between individual halos. We determine the convergence power spectrum as a function of the maximum halo mass and so provide the means to interpret results from surveys that lack high-mass halos either through selection criteria or small fields. Since shot noise from rare massive halos is mainly responsible for the sample variance below 10&arcmin;, our method should aid our ability to extract cosmological information from small fields.
Explore the source record for details and available documents.