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Biomedical subjects

L C Yuan

Publications and source records attributed to L C Yuan.

At least 19 recordsLinked to original sources
PubMed1996

Stability of cidofovir in 0.9% sodium chloride injection and in 5% dextrose injection.

The stability of cidofovir in i.v. admixtures under refrigerator and room temperature conditions was studied. Admixtures of cidofovir 0.21 and 8.12 mg/mL in 0.9% sodium chloride injection or 5% dextrose injection and of 0.085 and 3.51 mg/mL in 5% dextrose and 0.45% sodium chloride injection were prepared in triplicate in polyvinyl chloride (PVC) or polyethylene-polypropylene containers and i.v. administration sets and stored for 24 hours at 2-8 or 30 degrees C. The lower concentration of cidofovir corresponded to an assumed dose of 0.5 mg/kg for a 40-kg patient, and the higher concentration to an assumed dose of 10 mg/kg for a 100-kg patient. Samples were removed at 0 and 24 hours and analyzed for cidofovir concentration by high-performance liquid chromatography. Physical compatibility was also studied. The stability of cidofovir in 0.9% sodium chloride injection and in 5% dextrose injection at low- and high-dose concentrations was unaffected by storage at either temperature. All admixtures were clear, colorless, and free of visible particles or precipitation. There were no substantial changes in pH or number of particles of > or = 10 microns in diameter. Cidofovir 0.21 and 0.12 mg/mL was stable in 0.9% sodium chloride injection and 5% dextrose injection in PVC and polyethylene-polypropylene containers and i.v. administration sets for up to 24 hours at 2-8 and 30 degrees C. Cidofovir was compatible with the injectable solutions studied.

Antiviral Agents↗
PubMed1993

Uncleaved signals for glycosylphosphatidylinositol anchoring cause retention of precursor proteins in the endoplasmic reticulum.

Glycosylphosphatidylinositol (GPI)-anchored proteins are generally absent from the surface of cells that are defective in GPI biosynthesis. The current study was undertaken to: (a) examine in detail the intracellular localization and fate of precursors of GPI-anchored proteins in cells that fail to add GPI groups and (b) define structural characteristics of the precursor proteins that determine their intracellular localization. By examining GPI-deficient cells, we show that the uncleaved precursor of the GPI-anchored protein, Q7b, is retained in the cisternae of the endoplasmic reticulum (ER) and is largely lost intracellularly with a half-time of 2-4 h. Only a small amount (1-10%) of a proteolytically cleaved form of the protein is secreted into the medium. In cells competent for GPI anchor addition, mutation of the putative cleavage/attachment site for GPI addition in Q7b results in a similar phenotype of ER retention of the uncleaved precursor. An aspartic acid residue (Asp316) within the Q7b GPI anchoring signal, previously found to be essential for GPI anchor addition (Waneck, G. L., Stein, M. E., and Flavell, R. A. (1988) Science 241, 697-699), is also shown to be critical for ER retention. Information leading to ER retention is transferable to another protein leading to ER retention is transferable to another protein by fusion of the GPI anchoring signals from either Q7b or the GPI-anchored form of the IgG Fc receptor type III. Analysis by sedimentation on sucrose gradients shows that Q7b species retained in the ER are multimeric, whereas species that exit the ER are monomeric. This correlation suggests that the presence of an uncleaved signal for GPI anchoring induces changes in the aggregation state of the precursor proteins, which may lead to their retention in the ER.

Amino Acid Sequence↗
PubMed1993

Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence.

Protein localization to the TGN was investigated by examining the subcellular distribution of chimeric proteins in which the cytoplasmic and/or transmembrane domains of the TGN protein, TGN38, were substituted for the analogous domains of the plasma membrane protein, Tac. Using immunofluorescence and immunoelectron microscopy, the COOH-terminal cytoplasmic domain of TGN38 was found to be sufficient for localization of the chimeric proteins to the TGN. Deletion analysis identified an 11-amino acid segment containing the critical sequence, YQRL, as being sufficient for TGN localization. TGN localization was abrogated by mutation of the tyrosine or leucine residues in this sequence to alanine, or of the arginine residue to aspartate. In addition to specifying TGN localization, the 11-amino acid segment was active as an internalization signal, although the property of internalization alone was insufficient to confer TGN localization. Overexpression of chimeric proteins containing TGN localization determinants resulted in their detection at the plasma membrane and in intracellular vesicles, and abolished detection of endogenous TGN38. These results suggest that discrete cytoplasmic determinants can mediate protein localization to the TGN, and reveal a novel role for tyrosine-based motifs in this process.

Amino Acid Sequence↗
PubMed1992

Characterization of the heterogeneity of polyethylene glycol-modified superoxide dismutase by chromatographic and electrophoretic techniques.

Covalent attachment of polyethylene glycol (PEG) chains to the enzyme Cu,Zn-superoxide dismutase (SOD) produces a heterogeneous mixture of modified protein species. The heterogeneity of the product (PEG-SOD) derives from a variable stoichiometric combination of PEG with individual SOD molecules in addition to the polydispersity of the PEG reagent. Characterization of PEG-SOD presents significant challenges due in part to this heterogeneity in addition to the hybrid nature of the modified enzyme. The application of classical methods of protein characterization is not always successful for these PEG-proteins requiring the development of alternative or modified procedures. A series of chromatographic techniques including reversed-phase, ion-exchange, size-exclusion, and hydrophobic interaction high-performance liquid chromatography along with electrophoretic techniques including isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and capillary zone electrophoresis have been developed for assessing the degree of heterogeneity of PEG-SOD samples which encompass a range of different stoichiometries. Examples will be given demonstrating the application of these techniques to characterize PEG-SOD samples of different composition produced during the course of the reaction between SOD and an activated PEG reagent.

Animals↗
PubMed1991

A recycling pathway between the endoplasmic reticulum and the Golgi apparatus for retention of unassembled MHC class I molecules.

Assembly of class I major histocompatibility complex (MHC) molecules involves the interaction of two distinct polypeptides (the heavy and light chains) with peptide antigen. Cell lines synthesizing both chains but expressing low levels of MHC class I molecules on their surface as a result of a failure in assembly and transport have been identified. We now report that although the apparent steady-state distribution in these cells of class I molecules is in the endoplasmic reticulum (ER), the molecules in fact are recycled between the ER and Golgi, rather than retained in the ER. This explains the failure of class I molecules to negotiate the secretory pathway. Class I molecules do not seem to be modified by Golgi enzymes, suggesting that the proteins do not reach the Golgi apparatus during recycling. But morphological and subcellular fractionation evidence indicates that they pass through the cis Golgi or a Golgi-associated organelle, which we postulate to be the recycling organelle. This compartment, which we call the 'cis-Golgi network', would thereby be a sorting organelle that selects proteins for return to the ER.

Animals↗
PubMed1991

Novel redistribution of an intracellular pool of CD45 accompanies T cell activation.

The major tyrosine phosphatase activity against angiotensin detected in membranes of the antigen-specific T cell hybridoma 2B4 is contained in the cytoplasmic tail of the CD45 molecule. When these cells are stimulated with either an antibody directed against the T cell antigen receptor or an activating anti-Thy-1 antibody, there is a rapid redistribution of CD45 in the cells. The redistribution can be observed in two ways: morphology and subcellular fractionation. Morphologic examination of resting cells reveals intense CD45 staining of the Golgi as well as surface staining. Upon activation the Golgi is rapidly cleared of CD45. This redistribution is specific for CD45 and is not observed for an intrinsic Golgi protein, mannosidase II, or a protein traversing the secretory pathway, the T cell receptor. In activated cells, in contrast to resting cells, approximately 30% of the total cellular CD45 is precipitated either at 280 x g or at 200,000 x g through a 2.2 M sucrose cushion after cell homogenization. This fraction is not accessible to cell surface labeling. CD45 redistribution does not require hydrolysis of phosphatidylinositides and cannot be reproduced by the addition of phorbol ester and calcium ionophore. It does require the presence of an intact functional T cell receptor on the cell surface. These studies suggest that the residence time of CD45 within an intracellular organelle can be acutely regulated by a signal mediated via the T cell receptor. This regulation may control access of this phosphatase to critical substrates.

Animals↗
PubMed1990

Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway.

Characteristics of brefeldin A (BFA)-induced redistribution of Golgi proteins into the endoplasmic reticulum (ER) and its relationship to an ER retrieval pathway were investigated. Retrograde movement of Golgi proteins into the ER occurred via long, tubulovesicular processes extending out of the Golgi along microtubules. Microtubule-disrupting agents (i.e., nocodazole), energy poisons, and reduced temperatures inhibited this pathway. In BFA-treated cells Golgi proteins appeared to cycle between the ER and an intermediate compartment marked by a 53 kd protein. Addition of nocodazole disrupted this dynamic cycle by preferentially inhibiting retrograde movement, causing Golgi proteins to accumulate in the intermediate compartment. In the absence of BFA, such an ER cycling pathway appeared to be followed normally by the 53 kd protein but not by Golgi proteins, as revealed by temperature shift experiments. We propose that BFA induces the interaction of the Golgi with an intermediate "recycling" compartment that utilizes a microtubule-dependent pathway into the ER.

Animals↗
PubMed1989

Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER.

In cells treated with brefeldin A (BFA), movement of newly synthesized membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus was blocked. Surprisingly, the glycoproteins retained in the ER were rapidly processed by cis/medial Golgi enzymes but not by trans Golgi enzymes. An explanation for these observations was provided from morphological studies at both the light and electron microscopic levels using markers for the cis/medial and trans Golgi. They revealed a rapid and dramatic redistribution to the ER of components of the cis/medial but not the trans Golgi in response to treatment with BFA. Upon removal of BFA, the morphology of the Golgi apparatus was rapidly reestablished and proteins normally transported out of the ER were efficiently and rapidly sorted to their final destinations. These results suggest that BFA disrupts a dynamic membrane-recycling pathway between the ER and cis/medial Golgi, effectively blocking membrane transport out of but not back to the ER.

Animals↗
PubMed1988

Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins.

We have characterized a pre-Golgi, proteolytic pathway for rapid degradation of newly synthesized T cell receptor (TCR) subunits which is insensitive to drugs that block lysosomal proteolysis. The site of degradation in this pathway is either part of or closely related to the endoplasmic reticulum (ER). This "ER" degradative pathway very likely plays an important role in many cells in the removal of unassembled or incompletely assembled membrane protein complexes from the secretory pathway. It is the sole pathway followed by TCR alpha chains and alpha-beta complexes in transfected fibroblasts. In T cells treated with ionophores, which disrupt transport of the TCR from the ER to the Golgi, all newly synthesized alpha, beta, and delta chains are destroyed by this pathway. A variety of biochemical and morphological techniques have been used to distinguish the "ER" degradative pathway from an alternative, lysosomal pathway.

Animals↗
PubMed1988

Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment.

We have examined the fate of newly synthesized T cell antigen receptor (TCR) subunits in a T cell hybridoma deficient in expression of the clonotypic beta chain. Synthesis and assembly of the remaining chains proceed normally but surface expression of TCR chains is undetectable in these cells. A variety of biochemical and morphological techniques has been used to show that the TCR chains in these cells fail to be transported to any of the Golgi cisternae. Instead, they are retained in a pre-Golgi compartment which is either part of or closely related to the endoplasmic reticulum. The CD3-delta chain is degraded by a non-lysosomal process that is inhibited at temperatures at or below 27 degrees C. By contrast, the remaining chains (CD3-epsilon, CD3-gamma, and zeta) are very stable over 7 h. We propose possible mechanisms that may explain the differential fate of TCR chains retained in a pre-Golgi compartment.

Animals↗
PubMed1985

Cortical reaction and zona hardening in mouse oocytes following exposure to ethanol.

Mouse oocytes were treated with 8% ethanol for 3-6 min. The rate and pathways of parthenogenetic activation, occurrence of cortical reaction, and zona solubility changes were assessed in alcohol-treated eggs. The incidence of parthenogenetic activation was greatest (91%) after 3-4-min exposure, and it was reduced (84%) after 5-6-min exposure to alcohol. Also, the rate of haploid single pronucleate parthenogenones decreased and the rate of fragmented ova increased with increase time of exposure to ethanol. Ultrastructural observations showed occurrence of cortical reaction, disappearance and subsequent reappearance of short microvilli. A slight damage occurred to the ER in alcohol-exposed ova. The zona dissolution assay utilizing alpha-chymotrypsin demonstrated decreased solubility of the zonae pellucidae after exposure to alcohol. The zona dissolution t50 increased from 0.5-2.5 min in nontreated unfertilized oocytes to about 4 h in activated ova. The t50 of in vivo fertilized eggs was 4 1/2 h. Empty zonae exposed to alcohol lysed at the same rate as nontreated control zonae did. The results indicate that activation of mouse oocytes with alcohol initiates completion of meiosis and triggers the cortical reaction, which results in subsequent hardening of the zona pellucida.

Animals↗
PubMed1984

Use of riboflavin-binding protein to investigate steric and electronic relationships in flavin analogs and models.

We have examined the affinity of two recently synthesized flavin analogs for the isoalloxazine binding site of riboflavin-binding protein (RBP). The results showed that pyrimidopteridines could bind to RBP (Kd 160-250 microM). This suggested that, at the FMN or FAD level, these analogs might also bind to other apoflavoproteins, thereby providing a high potential probe for flavin enzymology. In contrast, 4a,5-ring-opened isoalloxazines did not bind to RBP. However, 1,10a-ring-opened flavins bind with considerable avidity (Kd about 40 nM). Evidence is presented which indicates that the 4a,5-ring-opened species adopted a nonplanar configuration which, in turn, was responsible for the lack of affinity to RBP. Steric and electronic consequences of a 4a,5 ring opening are discussed in relation to flavin-dependent phenolic hydroxylases.

Binding Sites↗
PubMed1983

Response of monkeys to porcine zona pellucida as detected by a solid-phase radioimmunoassay.

The immunological response of cynomolgus monkeys (Macaca fascicularis) to immunization was evaluated utilizing collagenase-isolated pig zona pellucida. Six weeks after initial immunization a high serum titer of antibody was exhibited. Serum antibody titers demonstrated a noticeable decline 5 months after booster injections were discontinued. The assay method used is rapid and is capable of detecting antibody in serum dilutions of 1:78,000, as compared to 1:125,000 with the indirect fluorescence assay.

Aluminum Hydroxide↗
PubMed1982

Identification of mammalian sperm surface antigens: II. Characterization of an acrosomal cap protein and a tail protein using monoclonal anti-mouse sperm antibodies.

Monoclonal anti-mouse sperm antibodies have been produced by fusing mouse myeloma cells with spleen cells from rats immunized with epididymal sperm of C3H mice, Immunoprecipitation and immunoperoxidase techniques showed that one such monoclonal antibody, AMS IV-33, recognized a 200 000 dalton protein localized on the acrosomal cap of the sperm cell. Two other monoclonal antibodies AMS IV-54 and -76, reacted with a 68 000 dalton component on the surface of the sperm tail. Both antigenic targets were species specific and were present in about equal amounts on sperm from several different strains of mice. The tail protein was sperm specific, whereas the antibody reacting with the acrosomal cap protein also appeared to react somewhat with antigens present in other mouse tissues.

Acrosome↗
PubMed1982

Identification of mammalian sperm surface antigens. I. Production of monoclonal anti-mouse sperm antibodies.

Surface antigens of mammalian sperm were studied by use of monoclonal antibodies (MAs). Six hybridoma cell lines were obtained by fusion of mouse myeloma cells with spleen cells from rats immunized with unwashed, epididymal sperm from C3H mice. Quantitative assessment of antibody binding, using a solid phase, antibody-protein A assay, indicated that four MAs bound to integral, sperm surface antigens; two others bound to nonintegral sperm antigens or epididymal fluid components. Immunofluorescence studies showed specific binding of individual MAs to localized regions: acrosome, midpiece, and midpiece and tail. All of these MAs inhibited sperm-egg binding, and those to the midpiece and/or tail immobilized sperm cells. The monoclonal antibodies provide probes for immunochemical characterization of sperm antigens and for elucidation of the role of the antigens in sperm.

Animals↗
PubMed1981

An improved method for processing single cells for electron microscopy utilizing agarose.

An improved method is presented for processing single cells for electron microscopy. Agarose, which has a low (30 degrees C) gelling temperature, was used as an initial embedding medium for single cells (spermatozoa and oocytes) and dissociated cell preparations (luteal cells and spleen cells). Dispersed cells of corpus luteum, spleen, and epididymal spermatozoa were placed in 1.5% agarose after aldehyde fixation. These fixed cells, embedded in agarose, were packed into a dense pellet by centrifugation, postfixed, then embedded in Epon. Mammalian eggs were not centrifuged; instead, they were embedded in agarose discs. Cells embedded in agarose were cooled below 30 degrees C to allow for gelling, then processed for electron microscopy. Because agarose has a low gelling temperature, some heat-labile substances were preserved, as demonstrated by retention of peroxidase activity using the DAB histochemical method. The agarose embedding procedure is both rapid and facile, and has proven to be of value in the handling of fragile single cells for electron microscopic studies.

Animals↗