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Biomedical subjects

Y S Cheng

Publications and source records attributed to Y S Cheng.

132 records · Page 8Linked to original sources
PubMedDate not supplied

Amino acid conservation and clinical severity of human glucose-6-phosphate dehydrogenase mutations.

More than a hundred naturally occurring mutations of human glucose-6-phosphate dehydrogenase (G6PD) have been identified at the amino acid level. The abundance of distinct mutation sites and their clinical manifestations make this enzyme ideal for structure-function analysis studies. We present here a sequence and structure combined analysis by which the severity of clinical symptoms resulting from point mutations of this enzyme is correlated with quantified degrees of amino acid conservation within 23 G6PD sequences from different organisms. Our analysis verifies, on a quantitative basis, a widely held notion that clinically severer mutations of G6PD usually occur at conserved amino acids. However, marked exceptions to this general trend exist which are most notably revealed by a number of mutations associated with chronic nonspherocytic hemolytic anemia (class I variants). When mapped onto a homology-derived structural model of human G6PD, these class I mutational sites of low amino acid conservation appear to localize in two spatially distinct clusters, both of which are populated with mutations consisting mainly of clinically severer variants (i.e. class I and class II). These results of computer-assisted analyses contribute to a further understanding of the structure-function relationships of human G6PD deficiency.

Amino Acid Sequence↗
PubMedDate not supplied

Synthesis and in vitro evaluation of [Leu13]porcine motilin fragments.

Several peptide fragments representing N-terminal, C-terminal, and internal sequences of [Leu13]porcine motilin ([Leu13]pMOT) were synthesized using Fmoc solid phase methodology. Peptides were assayed for motilin receptor binding activity in a rabbit antrum smooth muscle preparation and for stimulation of contractile activity in segments of rabbit duodenum. In vitro activity was directly correlated with motilin receptor binding affinity for all [Leu13]pMOT fragments examined. N-Terminal fragments of just over half the length of the native peptide are nearly equipotent as full-length motilin. These results suggest that the N-terminal segment, together with residues from the mid-portion of the molecule, constitutes the bioactive portion of pMOT. The C-terminal segment, in contrast, contributes little to receptor binding affinity or in vitro activity.

Amino Acids↗
PubMedDate not supplied

Stabilization of a degradable protein by its overexpression in Escherichia coli.

Synthesis of proteins in Escherichia coli using recombinant DNA methodology has become an important tool for isolating and studying proteins. However, the E. coli protein degradation systems can interfere with the expression of cloned genes. To examine the effect of protein degradation, we have cloned the X90 allele of the E. coli lacZ gene. The X90 allele, an ochre mutant, codes for beta-galactosidase lacking approx. 12 amino acids from the carboxyl terminus. The X90 protein is rapidly degraded in wild-type E. coli. Randomly sheared DNA fragments from lambda placZ-X90 were inserted into the EcoRI site of the plasmid pOP203-UV5-3, a derivative of pMB9 containing the lactose operator-promoter region. Recombinant plasmids that carry the lacZ-X90 gene were identified by the Lac+ phenotype of their transformants in an ochre-suppressor-containing host and the Lac- phenotype in Su degrees or supE hosts. One recombinant plasmid, p41, with an insert of 7.6 kb codes for the synthesis of the X90 promoter at a quantity equal to or greater than 50% of the total cellular protein of several strains. In contrast to the normal situation, the X90 molecules synthesized in great excess from the plasmid are stable in Su degrees hosts and can be recovered primarily from the 10 000 X g pellets of sonication lysates. The surprising stability of the overproduced X90 protein may be due to the formation of proteinaceous aggregates.

Bacterial Proteins↗
PubMedDate not supplied

Separation of pure and immunoreactive virus-like particles using gel filtration chromatography following immobilized metal ion affinity chromatography.

A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of the infectious bursal disease virus (IBDV) with six additional histidine residues at its C-terminus. The ultimate goal was the development of an efficient subunit vaccine against IBDV infection. The particles within the infected High-Five (Hi-5) cell lysates were partially purified by employing immobilized metal ion (Ni(2+)) affinity chromatography (IMAC). The initial step could recover approximately 85% of immunoreactive rVP2H proteins but failed to separate the rVP2H particles from the free rVP2H proteins or its degraded products. To separate the particulate form from the free form of rVP2H, an additional step was added, which used either gel filtration chromatography or CsCl density gradient ultracentrifugation. Both were able to produce extremely pure rVP2H particles with a buoyant density close to 1.27 g/cm(3). However, the former method can process a larger sample volume than does the latter. By integrating IMAC and gel filtration chromatography, 1 mg of extremely pure rVP2H particles was routinely obtained from a 500 mL Hi-5 cell culture broth. The separation of the particulate form from the free form of rVP2H proteins exposes their respective immunogenicity to induce the virus-neutralizing antibodies and the ability to protect chickens from IBDV infection. Additionally, the abundant quantities of pure rVP2H particles coupled with their uniform dimensions facilitates an understanding of higher order structure of the immunogenic particles and can therefore result in improved vaccines against the virus.

Baculoviridae↗
PubMedDate not supplied

The determination of biurea in the presence of azodicarbonamide by HPLC.

Azodicarbonamide (ADA), a solid blowing agent used in the manufacture of plastics, has been selected for inhalation toxicity testing by the National Toxicology Program. To test for decomposition of ADA during aerosolization, an HPLC method was developed to quantitate the relative amounts of one possible degradation product, biurea, in bulk samples and filter samples collected after aerosolization. The method uses a C18 column with 10-micron particles, UV monitoring at 190 nm for biurea and 425 nm for ADA, and a mobile phase of 100% water. Quantitation is with 14C-labeled biurea and ADA as external standards. The assay was validated by spiking bulk ADA with projected levels of 1, 2, and 3% biurea. Levels of biurea bound in both bulk and filter-collected aerosol samples of ADA were both 0.50%, with relative standard deviations of 13 and 26%, respectively.

Animals↗