Biomedical subjects

C Y Chiu

Publications and source records attributed to C Y Chiu.

At least 19 recordsLinked to original sources

PubMed1999

A helper-independent adenovirus vector with E1, E3, and fiber deleted: structure and infectivity of fiberless particles.

The adenovirus (Ad) fiber protein largely determines viral tropism through interaction with specific cell surface receptors. This molecule may also be involved in virion assembly or maturation, as some previously characterized fiber mutants were defective for processing of viral structural proteins. We previously described packaging cell lines that express Ad type 5 (Ad5) fiber and can complement the temperature-sensitive Ad fiber mutant H5ts142. We have now used these packaging cells to construct a new adenoviral vector (Ad5.betagal.DeltaF) with E1, E3, and L5 (fiber) deleted and analyzed the fiber null phenotype. Ad5.betagal.DeltaF growth was completely helper independent, and fiberless particles were produced by a single final round of growth in 293 cells. Cryoelectron microscopic studies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the structure and composition of these particles was nearly identical to those of first-generation Ad vectors. As expected, fiberless particles had reduced infectivity on epithelial cells, but they retained the ability to infect monocytic cells via an integrin-dependent pathway. These studies provide a novel approach to developing retargeted Ad gene therapy vectors.

Adenovirus E1 Proteins↗

PubMed1998

Cryo-EM imaging of the catalytic subunit of the DNA-dependent protein kinase.

The DNA-dependent protein kinase (DNA-PK) plays an important role in mammalian DNA double-strand break repair and immunoglobulin gene rearrangement. The DNA-PK holoenzyme is activated by assembly at DNA ends and is comprised of DNA-PKcs, a 460 kDa protein kinase catalytic subunit, and Ku, a 70 kDa/80 kDa heterodimeric DNA-targeting component. We have solved the three-dimensional structure of DNA-PKcs to approximately 21 A resolution by analytically combining images of nearly 9500 individual particles extracted from cryo-electron micrographs. The DNA-PKcs protein has an open, pseudo 2-fold symmetric structure with a gap separating a crown-shaped top from a rounded base. Columns of density are observed to protrude into the gap from both the crown and the base. Measurements of the enclosed volume indicate that the interior of the protein is largely hollow. The structure of DNA-PKcs suggests that its association with DNA may involve the internalization of double-stranded ends.

Cryoelectron Microscopy↗

PubMed1998

Modification of a receptor-binding surface of epidermal growth factor (EGF): analogs with enhanced receptor affinity at low pH or at neutrality.

Six mutants of human epidermal growth factor (EGF), which carry single point substitutions within a surface patch proposed to juxtapose the bound receptor, were prepared and characterized for receptor affinity and mitogenicity. Receptor affinities relative to EGF are G12Q > H16D > Y13W > Q43A approximately = H16A approximately = EGF >> L15A. Notably, the reduced receptor affinity of mutant L15A indicates that Leu15 probably contributes substantially to receptor binding whereas unaltered receptor affinities observed for analogs H16A and Q43A indicate that neither His16 nor Gln43 contributes significantly to this interaction. On the other hand, the observed enhanced receptor affinities of analogs G12Q, Y13W and H16D highlight surface loci where additional productive receptor-binding contacts can be introduced. Interestingly, at acidic pH analog H16A reveals substantially greater receptor affinity than that of EGF, a property which may offer enhanced therapeutic utility in acidic environments in vivo.

Amino Acid Sequence↗

PubMed1997

Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization.

Interaction of the adenovirus penton base protein with alpha v integrins promotes virus entry into host cells. The location of the integrin binding sequence Arg-Gly-Asp (RGD) on human type 2 adenovirus (Ad2) was visualized by cryo-electron microscopy (cryo-EM) and image reconstruction using a mAb (DAV-1) which recognizes a linear epitope, IRGDTFATR. The sites for DAV-1 binding corresponded to the weak density above each of the five 22 A protrusions on the adenovirus penton base protein. Modeling of a Fab fragment crystal structure into the adenovirus-Fab cryo-EM density indicated a large amplitude of motion for the Fab and the RGD epitope. An unexpected finding was that Fab fragments, but not IgG antibody molecules, inhibited adenovirus infection. Steric hindrance from the adenovirus fiber and a few bound IgG molecules, as well as epitope mobility, most likely prevent binding of IgG antibodies to all five RGD sites on the penton base protein within the intact virus. These studies indicate that the structure of the adenovirus particle facilitates interaction with cell integrins, whilst restricting binding of potentially neutralizing antibodies.

Adenoviridae↗

PubMed1997

Phosphorothioate oligodeoxynucleotides: large-scale synthesis and analysis, impurity characterization, and the effects of phosphorus stereochemistry.

Large-scale synthesis of phosphorothioate oligodeoxynucleotides on Tentagel using a 'batch mode' synthesizer and beta-cyanoethyl phosphoramidite coupling followed by sulfurization with bis(O,O-diisopropoxy phosphinothioyl) disulfide (S-tetra) provides stepwise yields of 98-99% and results in phosphorothioate oligodeoxynucleotides that are 93-97% pure, as determined by PAGE, after reverse-phase high performance liquid chromatography (RP-HPLC) and 'downstream' processing. The purity of phosphorothioate oligodeoxynucleotides synthesized on Tentagel is significantly higher than those synthesized on controlled pore glass. Electrospray ionization mass spectrometry of the n-1 impurity isolated by preparative PAGE was used to establish that the n-1 impurity is a heterogeneous mixture of all possible single-deletion sequences, relative to the parent phosphorothioate oligodeoxynucleotide, and results from minor, though repetitive, imperfections in the synthesis cycle. Acid-catalysed depurination was found to occur both during the synthesis and during the post-synthesis detritylation, following RP-HPLC. Studies of hybridization affinity and biological mechanism of action using independently synthesized n-1 phosphorothioate oligodeoxynucleotides relative to the 15 mer LR-3280 showed that, in this case, the majority of the n-1 sequences had more than a 10 degrees C decrease in melting temperature with sense RNA compared to the n-mer, and they did not cause detectable cleavage of RNA by RNase H in HL-60 human promyelocytic leukaemia cells. P stereoregular phosphorothioate oligodeoxynucleotides are not significantly more active than their stereorandom counterparts and thus their use in clinical studies seems unwarranted.

Humans↗

PubMed1997

Lay dispositionism and implicit theories of personality.

Lay dispositionism refers to lay people's tendency to use traits as the basic unit of analysis in social perception (L. Ross & R. E. Nisbett, 1991). Five studies explored the relation between the practices indicative of lay dispositionism and people's implicit theories about the nature of personal attributes. As predicted, compared with those who believed that personal attributes are malleable (incremental theorists), those who believed in fixed traits (entity theorists) used traits or trait-relevant information to make stronger future behavioral predictions (Studies 1 and 2) and made stronger trait inferences from behavior (Study 3). Moreover, the relation between implicit theories and lay dispositionism was found in both the United States (a more individualistic culture) and Hong Kong (a more collectivistic culture), suggesting this relation to be generalizable across cultures (Study 4). Finally, an experiment in which implicit theories were manipulated provided preliminary evidence for the possible causal role of implicit theories in lay dispositionism (Study 5).

Female↗

PubMed1996

Safety and immunogenicity of a serogroups A/C Neisseria meningitidis oligosaccharide-protein conjugate vaccine in young children. A randomized controlled trial.

OBJECTIVE: To assess the safety and immunogenicity of a bivalent serogroups A/C meningococcal oligosaccharide-protein conjugate vaccine compared with the licensed meningococcal polysaccharide vaccine. DESIGN: Randomized controlled trial. STUDY POPULATION: Ninety healthy 18- to 24-month-old children who were seen at a southern California Kaiser Permanente clinic. INTERVENTIONS: Vaccination with either the meningococcal conjugate vaccine (at 1 of 2 dosages) or the polysaccharide vaccine, with 2 doses given 2 months apart. MAIN OUTCOME MEASUREMENTS: Immune response to each vaccine dose as determined by measurement of serogroup-specific total antibodies by enzyme-linked immunosorbent assay (ELISA) and by assessment of serum bactericidal activity. RESULTS: Both vaccines appeared to be safe, and nearly all children responded with greater than 4-fold increases in antibody levels. The 2 dosages of the conjugate vaccine induced similar antibody responses; therefore, the data for the 2 conjugate vaccine groups were combined. Following 2 doses, ELISA antibody levels against group C meningococcus were significantly higher in conjugate vaccine recipients than in polysaccharide vaccine recipients (16.66 microg/mL vs. 8.31 microgm/mL; P<.001), but antibody levels against group A were not significantly different 22.75 microg/mL vs 21.24 microg/mL; P=.70). The serum bactericidal assays showed striking differences between the conjugate and polysaccharide vaccine groups. Geometric mean serum bactericidal titers were significantly higher in conjugate vaccine recipients (755.6 vs 37.6 for group A, P<.001; 3197.9 vs 11.4 for group C, P<.001). CONCLUSIONS: The immune response induced by this meningococcal oligosaccharide-protein conjugate vaccine was qualitatively different from that induced by the polysaccharide vaccine, and the antibodies it elicited provided greater functional activity.

Antibodies, Bacterial↗

PubMed1996

Oligonucleotide N3'-->P5' phosphoramidates as antisense agents.

Uniformly modified oligonucleotide N3'-->P5' phosphoramidates, where every 3'-oxygen is replaced by a 3'-amino group, were synthesized. These compounds have very high affinity to single-stranded RNAs and thus have potential utility as antisense agents. As was shown in this study, the oligonucleotide phosphoramidates are resistant to digestion with snake venom phosphodiesterase, to nuclease activity in a HeLa cell nuclear extract, or to nuclease activity in 50% human plasma, where no significant hydrolysis was observed after 8 h. These compounds were used in various in vitro cellular systems as antisense compounds addressed to different targeted regions of c-myb, c-myc and bcr-abl mRNAs. C-myb antisense phosphoramidates at 5 microM caused sequence and dose-dependent inhibition of HL-60 cell proliferation and a 75% reduction in c-myb protein and RNA levels, as determined by Western blot and RT-PCR analysis. Analogous results were observed for anti-c-myc phosphoramidates, where a complete cytostatic effect for HL-60 cells was observed at 1 microM concentration for fully complementary, but not for mismatched compounds, which were indistinguishable from untreated controls. This was correlated with a 93% reduction in c-myc protein level. Moreover, colony formation by the primary CML cells was also inhibited 75-95% and up to 99% by anti-c-myc and anti-bcr-abl phosphoramidate oligonucleotides, respectively, in a sequence- and dose-dependent manner within a 0.5 nM-5 microM dose range. At these concentrations the colony-forming ability of normal bone marrow cells was not affected. The presented in vitro data indicate that oligonucleotide N3'-->P5' phosphoramidates could be used as specific and efficient antisense agents.

Animals↗

PubMed1996

Safety and immunogenicity of a recombinant hepatitis B vaccine administered to infants at 2, 4 and 6 months of age. The Kaiser-UCLA Vaccine Study Group.

A recombinant hepatitis B vaccine was administered to over 5000 infants in a prospective, randomized and blinded study. Infants were given either recombinant hepatitis B vaccine (Engerix-B, SmithKline Beecham Pharmaceuticals, 10 micrograms dose-1) or a Haemophilus influenzae type b (Hib) conjugate vaccine at 2, 4 and 6 months of age simultaneously with diphtheria-tetanus-pertussis and oral polio vaccines. Adverse reactions were ascertained by parental reports and interviews, and review of medical records. Blood specimens collected from 269 infants given hepatitis B vaccine were assayed for antibody to hepatitis B surface antigen (anti-HBs) by enzyme immunoassay. Infants given hepatitis B vaccine experienced low rates of adverse reactions that were similar or lower than the rates in infants given Hib conjugate vaccine. The geometric mean anti-HBs concentrations were 9.6 mIU ml-1 after one dose, 333 mIU ml-1 after two doses and 1812 mIU ml-1 after three doses (99% had levels > or = 10 mIU ml-1). Antibody responses to diphtheria and tetanus toxoids were unaffected by simultaneous administration of hepatitis B or Hib conjugate vaccine. Engerix-B vaccine was safe and immunogenic when given with other routine childhood immunizations at 2, 4 and 6 months of age, and should provide long-term protection against hepatitis B virus infection.

Antibodies, Bacterial↗

PubMed1996

Comparative safety and immunogenicity of two recombinant hepatitis B vaccines given to infants at two, four and six months of age.

OBJECTIVES: To evaluate the relative safety and immunogenicity of the two recombinant hepatitis B vaccines licensed in the United States with doses recommended for routine immunization of low risk infants and a schedule that corresponds with routine pediatric visits. METHODS: Healthy infants were immunized at 2, 4 and 6 months of age with hepatitis B vaccine manufactured by either SmithKline Beecham (Engerix-B, 10 micrograms/dose, n = 228) or Merck and Co. (Recombivax HB, 2.5 micrograms/dose, n = 200). Adverse reactions were ascertained by parental reports and interviews and by review of medical records. Antibody concentrations to hepatitis B surface antigen (anti-HBs) were measured in sequential serum specimens by enzyme immunoassay. RESULTS: Adverse reactions were mild and the rates were not significantly different between the two groups. After the first and second doses the rates of seropositivity (> or = 10 mIU/ml) and seroprotection (> or = 10 mIU/ml) were significantly higher in infants given SmithKline Beecham vaccine (P < 0.01). After the second and third doses infants given SmithKline Beecham vaccine also had significantly higher geometric mean anti-HBs concentrations compared with those given Merck vaccine (348.0 mIU/ml vs. 66.9 and 1914.8 mIU/ml vs. 514.8 mIU/ml, respectively, P < 0.001). Nevertheless after the third dose 99% of infants in both vaccine groups achieved seroprotective antibody concentrations. CONCLUSIONS: Both recombinant hepatitis B vaccines were safe and immunogenic when administered concurrently with other pediatric vaccines at 2, 4 and 6 months of age, but earlier protective responses were observed with the SmithKline Beecham vaccine than with the Merck vaccine.

Consumer Product Safety↗

PubMed1995

Synthesis and characterization of a monomeric mutant Cu/Zn superoxide dismutase with partially reconstituted enzymic activity.

A monomeric analog of human Cu/Zn superoxide dismutase (F50E/G51E SOD), previously characterized and found to have reduced enzymic activity, was here further modified by replacing Glu133 with Gln. This substitution does not dramatically affect the coordination geometry at the active site, but enhances enzymic activity, and also increases the affinity for anions at the active site. This behavior parallels earlier published results in which this point mutation was made in the dimeric wild-type enzyme. The analog described here has afforded for the first time a monomeric superoxide dismutase with substantial activity. This point mutation does not significantly influence the protein structure but interactions with anions, including superoxide, are altered with respect to the monomeric form. The present monomeric Glu133Gln mutant has partially restored enzymic activity. The diminished activity of the monomeric analogs is discussed in the light of possible minor structural changes and some of their characteristics are compared with those of naturally occurring mutants associated with various neurological diseases.

Binding Sites↗

PubMed1995

Auditory priming for nonverbal information: Implicit and explicit memory for environmental sounds.

Three experiments examined repetition priming for meaningful environmental sounds (e.g., clock ticking, tooth brushing, toilet flushing, etc.) in a sound stem identification paradigm using brief sound cues. Prior encoding of target sounds together with their associated names facilitated subsequent identification of sound stems relative to nonstudied controls. In contrast, prior exposure to the names alone in the absence of the environmental sounds did not prime subsequent sound stem identification performance at all (Experiments 1 and 3). Explicit and implicit memory were dissociated such that sound stem cued recall was higher following semantic than nonsemantic encoding, whereas sound priming was insensitive to manipulations of depth of encoding (Experiments 2 and 3). These results extend the findings of long-term repetition priming into the auditory nonverbal domain and suggest that priming for environmental sounds is mediated primarily by perceptual processes.

Acoustic Stimulation↗

PubMed1995

High yields of interleukin-8 produced by a synthetic gene expressed in Escherichia coli and purified with a single antibody affinity column.

We developed a highly efficient expression system for the production of interleukin-8 (IL-8) in Escherichia coli. A synthetic gene used in the vector was designed to code for the 72-amino-acid form of IL-8 and incorporate additional new restriction sites. IL-8 was expressed in very large amounts in the periplasmic space and extracted by a gentle method which did not utilize denaturants. About 69% of the protein extracted from the periplasmic space was properly processed IL-8. A single anti-IL-8 monoclonal antibody affinity chromatography column yielded homogeneous IL-8 as determined by HPLC molecular sieve chromatography and amino-terminal sequencing. Between 14 and 22 mg of IL-8 was purified per liter of bacterial culture, in which the wet weight of E. coli was 7.6 g/liter. The recombinant IL-8 was fully active compared to published data and a commercially available preparation of recombinant IL-8. Our IL-8 and the commercial product had identical neutrophil binding isotherms, chemotactic activities, and enzyme release properties.

Amino Acid Sequence↗

PubMed1995

Effect of neonatal immunization with diphtheria and tetanus toxoids on antibody responses to Haemophilus influenzae type b conjugate vaccines.

We randomly assigned 150 newborn infants to receive diphtheria and tetanus toxoids (DT) or Hib oligosaccharide conjugate (HbOC) at birth to determine whether exposure to the Haemophilus influenzae type b (Hib) conjugate vaccines' carrier proteins would enhance immune responses to subsequent administrations of HbOC or PRP-tetanus toxoid conjugate (PRP-T) at 2, 4, and 6 months of age. Their antibody responses were compared with those of 100 children immunized with HbOC or PRP-T beginning at 2 months of age. No serious adverse reactions were associated with neonatal vaccination. Administration of HbOC at birth did not lead to earlier or higher antibody levels. Newborn immunization with DT did not prime children for enhanced antibody responses. Moreover, Hib antibody levels were lower in DT-primed children than in children immunized beginning at 2 months of age. Diphtheria antibody levels, but not tetanus antibody levels, were also lower in children immunized with DT at birth. We conclude that neonatal immunization with Hib conjugate vaccines is not a means to provide earlier protection against invasive Hib disease. Newborn DT administration does not enhance subsequent antibody responses to Hib conjugate vaccines, and may lead to suppression of Hib and diphtheria antibody responses.

Antibodies, Bacterial↗

PubMed1995

Enhanced antibody responses in infants given different sequences of heterogeneous Haemophilus influenzae type b conjugate vaccines.

To evaluate the safety and immunogenicity of differing sequences of heterogeneous Haemophilus influenzae type b (Hib) conjugate vaccines, we randomly assigned 300 infants to one of six vaccination schedules. At 2, 4, and 6 months of age, subjects were given single or heterogeneous vaccines: Hib polysaccharide (PRP) conjugated to mutant diphtheria toxin (HbOC), PRP conjugated to outer-membrane protein of Neisseria meningitidis (PRP-OMP), or PRP conjugated to tetanus toxoid (PRP-T). No serious reactions were attributable to immunization with heterogeneous vaccines, and there were few significant differences in the rates of minor adverse reactions among groups. PRP-OMP was the only vaccine that induced an antibody response after the first dose, but significant booster responses were not seen after the second and third doses. Subjects given PRP-T vaccine responded well after two doses, but three doses of HbOC vaccine were needed for an equivalent antibody response. All the Hib vaccine schedules evaluated were immunogenic, and schedules initiated by PRP-OMP vaccine at 2 months of age, followed by two doses of either HbOC or PRP-T vaccine at 4 and 6 months of age, induced the highest antibody levels after each dose. Such schedules may be the best for protecting infants and children who are at greatest risk of having invasive Hib disease, such as American Indian children.

Aging↗

PubMed1995

Yeast expression and phagemid display of the human urokinase plasminogen activator epidermal growth factor-like domain.

The human urokinase plasminogen activator (uPA) epidermal growth factor-like domain (residues 1-48) and a variant with a C-terminal epitope tag have been secreted from recombinant yeast. Purified human uPA 1-48 and uPA 1-48glu complete for binding to the human uPA receptor with Kds of 180 and 400 pM respectively, in an in vitro assay using an immobilized recombinant uPA receptor. A synthetic gene encoding human uPA 1-48 with an N-terminal epitope tag was inserted into a phagemid expression vector as a fusion with residues 249-406 of the M13 pIII protein with an intervening amber codon (TAG). Phagemid production led to infectious particles which were selectively bound and eluted from both epitope tag antibody and urokinase receptor. Sequential binding to this antibody and receptor demonstrated a substantial enrichment, where up to 10% of the infectious particles were then retained on urokinase receptor-coated plates. A PCR strategy was used to convert previously described peptide bacteriophage ligands for the urokinase receptor to phagemid display. The yields of these peptide phagemids and the uPA 1-48 phagemid showed a correlation with peptide affinity, in contrast to when the peptides are multivalently displayed on a bacteriophage.

Amino Acid Sequence↗

PubMed1994

A spectroscopic characterization of a monomeric analog of copper, zinc superoxide dismutase.

A mutated protein of human Cu(II)2Zn(II)2 SOD in which residues Phe50 and Gly51 at the dimer interface were substituted by Glu's, thus producing a monomeric species, has been characterized by electronic absorption spectroscopy, EPR, relaxivity and 1H NMR techniques. Such substitutions and/or accompanying remodeling and exposure of the dimer interface to solvent, alter the geometry of the active site: increases in the axiality of the copper chromophore and the Cu-OH2 distance have been observed. The affinity of both metal binding sites for Co(II) is also altered. The observed NMR parameters of the Co(II) substituted derivative have been interpreted as a function of the decrease of rotational correlation time as a consequence of the lower molecular weight of the mutated protein. Sharper NMR signals are also obtained for the reduced diamagnetic enzyme. Results are consistent with an active site structure similar to that observed for the dimeric analog. Thr137Ile characterized elsewhere. An observed proportional decrease in enzymatic activity and affinity for the N3-anion suggests the importance of electrostatic forces during substrate docking and catalysis.

Circular Dichroism↗

PubMed1994

Varieties of priming.

Recent data from brain-damaged and normal subjects converge to suggest several characteristics of repetition priming: firstly, it is sensitive to the physical and structural properties of input; secondly, it is unaffected by semantic processing at encoding; thirdly, it is frequently preserved in amnesic patients with impaired explicit memory; fourthly, it depends upon perceptual representations processed and stored by modality-specific cortical memory systems; and, finally, it is subject to constraints particular to the task employed and the type of information that is primed.

Auditory Perception↗