Biomedical subjects

Yudong Wang

Publications and source records attributed to Yudong Wang.

8 recordsLinked to original sources

PubMed2006

Pharmaceutical and analytical characterisation of (2R)-(3-amino-2-fluoropropyl)sulphinic acid, a GABAB receptor agonist.

(2R)-(3-amino-2-fluoropropyl)sulphinic acid (AFPSiA) is a potent GABAB agonist, which makes it a possible alternative in future GERD treatment. The degradation of AFPSiA was investigated to support the drug-development effort. The compound is too polar to be compatible with regular reversed-phase LC. Moreover, the compound and the degradation products cannot be detected by UV due to low absorption. Instead, the degradation of AFPSiA was followed by two different capillary electrophoresis methods with indirect UV detection and 1H NMR and 19F NMR. AFPSiA was very unstable in basic conditions and at temperatures above room temperature. The corresponding sulphonic acid and allylamine are formed via two separate degradation routes. Both these degradation products may cause unwanted side-effects in vivo. Aqueous solutions of AFPSiA were found to be more stable at pH between 1 and 3. It was suggested that AFPSiA should be stored frozen, preferably at -70 degrees C. In solid state, the compound can be stored at ambient conditions in closed vials with low relative humidity. A solid oral dosage form should be kept in a blister package.

Administration, Oral↗

PubMed2006

Profiling Caenorhabditis elegans non-coding RNA expression with a combined microarray.

Small non-coding RNAs (ncRNAs) are encoded by genes that function at the RNA level, and several hundred ncRNAs have been identified in various organisms. Here we describe an analysis of the small non-coding transcriptome of Caenorhabditis elegans, microRNAs excepted. As a substantial fraction of the ncRNAs is located in introns of protein-coding genes in C.elegans, we also analysed the relationship between ncRNA and host gene expression. To this end, we designed a combined microarray, which included probes against ncRNA as well as host gene mRNA transcripts. The microarray revealed pronounced differences in expression profiles, even among ncRNAs with housekeeping functions (e.g. snRNAs and snoRNAs), indicating distinct developmental regulation and stage-specific functions of a number of novel transcripts. Analysis of ncRNA-host mRNA relations showed that the expression of intronic ncRNA loci with conserved upstream motifs was not correlated to (and much higher than) expression levels of their host genes. Even promoter-less intronic ncRNA loci, though showing a clear correlation to host gene expression, appeared to have a surprising amount of 'expressional freedom', depending on host gene function. Taken together, our microarray analysis presents a more complete and detailed picture of a non-coding transcriptome than hitherto has been presented for any other multicellular organism.

Animals↗

PubMed2005

Organization of the Caenorhabditis elegans small non-coding transcriptome: genomic features, biogenesis, and expression.

Recent evidence points to considerable transcription occurring in non-protein-coding regions of eukaryote genomes. However, their lack of conservation and demonstrated function have created controversy over whether these transcripts are functional. Applying a novel cloning strategy, we have cloned 100 novel and 61 known or predicted Caenorhabditis elegans full-length ncRNAs. Studying the genomic environment and transcriptional characteristics have shown that two-thirds of all ncRNAs, including many intronic snoRNAs, are independently transcribed under the control of ncRNA-specific upstream promoter elements. Furthermore, the transcription levels of at least 60% of the ncRNAs vary with developmental stages. We identified two new classes of ncRNAs, stem-bulge RNAs (sbRNAs) and snRNA-like RNAs (snlRNAs), both featuring distinct internal motifs, secondary structures, upstream elements, and high and developmentally variable expression. Most of the novel ncRNAs are conserved in Caenorhabditis briggsae, but only one homolog was found outside the nematodes. Preliminary estimates indicate that the C. elegans transcriptome contains approximately 2700 small non-coding RNAs, potentially acting as regulatory elements in nematode development.

Animals↗

PubMed2004

Cadmium inhibits the electron transfer chain and induces reactive oxygen species.

Recent research indicates that cadmium (Cd) induces oxidative damage in cells; however, the mechanism of the oxidative stress induced by this metal is unclear. We investigated the effects of Cd on the individual complexes of the electron transfer chain (ETC) and on the stimulation of reactive oxygen species (ROS) production in mitochondria. The activity of complexes II (succinate:ubiquinone oxidoreductase) and III (ubiquinol:cytochrome c oxidoreductase) of mitochondrial ETC from liver, brain, and heart showed greater inhibition by Cd than the other complexes. Cd stimulated ROS production in the mitochondria of all three tissues mentioned above. The effect of various electron donors (NADH, succinate, and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol) on ROS production was tested separately in the presence and in the absence of Cd. ESR showed that complex III might be the only site of ROS production induced by Cd. The results of kinetic studies and electron turnover experiments suggest that Cd may bind between semiubiquinone and cytochrome b566 of the Q0 site of cytochrome b of complex III, resulting in accumulation of semiubiquinones at the Q0 site. The semiubiquinones, being unstable, are prone to transfer one electron to molecular oxygen to form superoxide, providing a possible mechanism for Cd-induced generation of ROS in mitochondria.

Animals↗

PubMed2003

Aspartate-186 in the head group of the yeast iron-sulfur protein of the cytochrome bc1 complex contributes to the protein conformation required for efficient electron transfer.

Two conserved charged amino acids, aspartate-186 and arginine-190, localized in the aqueous head region of the iron-sulfur protein of the cytochrome bc(1) complex of yeast mitochondria, were mutated to alanine, glutamate, or asparagine and isoleucine, respectively. The R190I mutation resulted in the complete loss of antimycin- and myxothiazol-sensitive cytochrome c reductase activity due to loss of more than 60% of the iron-sulfur protein in the complex. Mitochondria isolated from the D186A mutant had a 50% decrease in cytochrome c reductase activity but no loss of the iron-sulfur protein or the [2Fe-2S] cluster. The midpoint potential of the [2Fe-2S] cluster of the D186A mutant was decreased from 281 to 178 mV. The D186E and D186N mutations did not result in a loss of cytochrome c reductase activity or content of iron-sulfur protein; however, the redox potential of the [2Fe-2S] cluster of D186N was decreased from 281 to 241 mV. Molecular modeling/dynamics studies predicted that substituting an alanine for Asp-186 causes global structural changes in the head group of the iron-sulfur protein resulting in changes in the orientation of the [2Fe-2S] cluster and consequently a lowered redox potential. The rate of electrogenic proton pumping in the bc(1) complex isolated from mutant D186A reconstituted into proteoliposomes decreased 64%; however, the H(+)/2e(-) ratio of 1.9 was identical in the mutant and the wild-type complexes. The carboxyl binding reagent, N-(ethoxycarbonyl)-2-ethoxyl-1,2-dihydroquinoline (EEDQ) blocked electrogenic proton pumping in the bc(1) complex reconstituted into proteoliposomes without affecting electron transfer resulting in a decrease in the H(+)/2e(-) ratio to 1.2 and 1.1, respectively. EEDQ was bound to the iron-sulfur protein and core protein II in both the wild type and the D186A mutant, indicating that Asp-186 of the iron-sulfur protein is not required for proton translocation in the bc(1) complex.

Amino Acid Substitution↗

PubMed2003

Topographical organization of the N-terminal segment of lung pulmonary surfactant protein B (SP-B(1-25)) in phospholipid bilayers.

The location and depth of each residue of lung pulmonary surfactant protein B (SP-B(1-25)) in a phospholipid bilayer (PB) was determined by fluorescence quenching using synthesized single-residue-substituted peptides that were reconstituted into 1,2-dipalmitoyl phosphatidylcholine (DPPC)-enriched liposomes. The single-residue substitutions in peptides were either aspartate or tryptophan. The aspartate was subsequently labeled with the N-cyclohexyl-N'-(4-(dimethylamino)naphthyl)carbodiimide (NCD-4) fluorophore, whereas tryptophan is autofluorescent. Spin-labeled compounds, 5-doxylstearic acid (5-DSA), 7-doxylstearic acid (7-DSA), 12-doxylstearic acid (12-DSA), 4-(N,N-dimethyl-N-hexadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-16), and 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxy iodide (CAT-1), were used in the quenching experiments. The effective quenching order is determined by the accessibility of the quencher to a fluorescent group on the peptide. The order of quenching efficiency provides information about the relative locations of individual residues in the PB. Our data indicate that residues Phe1-Pro6 are located at the surface of PB, residues Tyr7-Trp9 are embedded in PB, and residues Leu10-Ile22 are involved in an amphipathic alpha-helix with its axis parallel to the surface of PB; residues Pro23-Gly25 reside at the surface. The effects of intermolecular disulfide bond formation in the SP-B(1-25) dimer were also investigated. The experiments suggest that the SP-B helix A has to rotate at an angle to form a disulfide bond with the neighboring cysteine, which makes the hydrophobic sides of the amphipathic helices face each other, thus forming a hydrophobic domain. The detailed topographical mapping of SP-B(1-25) and its dimer in PB provides new insights into the conformational organization of the lung pulmonary surfactant proteins in the environment that mimics the native state. The environment-specific conformational flexibility of the hydrophobic domain created by SP-B folding may explain the key functional properties of SP-B including their impact on phospholipid transport between the lipid phases and in modulating the cell inflammatory response during respiratory distress syndrome.

Amino Acid Sequence↗

PubMed2003

High-throughput screening of pKa values of pharmaceuticals by pressure-assisted capillary electrophoresis and mass spectrometry.

A high-throughput pKa screening method based on pressure-assisted capillary electrophoresis (CE) and mass spectrometry (MS) is presented. Effects of buffer type and ionic strength on sensitivity and pKa values were investigated. Influence of dimethyl sulfoxide (DMSO) concentration present in the sample on effective mobility measurement was examined. A series of ten volatile buffers, covering a pH range from 2.5 to 10.5 with the same ionic strength, was employed. The application of volatile background electrolytes resulted in significant signal increase as compared with commonly used non-volatile phosphate buffers. In general, the CE/MS system provided a ten-fold higher sensitivity than conventional UV detection. The newly developed CE/MS method offers high-throughput capacity by pooling a number of compounds into a single sample. Simultaneous measurement of more than 50 compounds was readily achieved in less than 150 min. The measured pKa values are consistent with the published data obtained from the CE/UV method and are also in good agreement with data generated by other methods. Other advantages of using CE/MS for pKa screening are illustrated with typical examples, including poorly soluble compounds and non-UV-absorbing compounds.

Algorithms↗

PubMed2002

Molecular modeling studies of the DCCD-treated cytochrome bc1 complex: predicted conformational changes and inhibition of proton translocation.

Dicyclohexylcarbodiimide (DCCD) binds covalently to an acidic amino acid located in the cd loop connecting membrane-spanning helices C and D of cytochrome b resulting in an inhibition of proton translocation in the cytochrome bc1 complex with minimal effects on the steady state rate of electron transfer. Single turnover studies performed with the yeast cytochrome bc1 complex indicated that the initial phase of cytochrome b reduction was inhibited 25-45% in the DCCD-treated cytochrome bc1 complex, while the rate of cytochrome c1 reduction was unaffected. Simulations by molecular modeling predict that binding of DCCD to glutamate 163 located in the cd2 loop of cytochrome b of chicken liver mitochondria results in major conformational changes in the protein. The conformation of the cd loop and the end of helix C appeared twisted with a concomitant rearrangement of the amino acid residues of both cd1 and cd2 loops. The predicted rearrangement of the amino acid residues of the cd loop results in disruptions of the hydrogen bonds predicted to form between amino acid residues of the cd and ef loops. Simultaneously, two new hydrogen bonds are predicted to form between glutamate 272 and two residues, aspartate 253 and tyrosine 272. Formation of these new hydrogen bonds would restrict the rotation and protonation of glutamate 272, which may be necessary for the release of the second electrogenic proton obtained during ubiquinol oxidation in the bc1 complex.

Binding Sites↗