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Biomedical subjects

P Chong

Publications and source records attributed to P Chong.

70 records · Page 4Linked to original sources
PubMed1991

A novel process for preparing an acellular pertussis vaccine composed of non-pyrogenic toxoids of pertussis toxin and filamentous hemagglutinin.

A novel process for preparing non-pyrogenic toxoids of pertussis toxin (PT) and filamentous hemagglutinin (FHA) is described. The process consists of chromatographies on perlite then on hydroxylapatite. Purification yields for PT and FHA are 62 and 68%, respectively. The purification process takes advantage of the novel use of perlite (a filter aid) for the simultaneous purification of PT and FHA. The hydroxylapatite, in addition to removing the remaining contaminants, also concentrates the antigens. The resulting PT and FHA are approximately 95% pure, and are non-pyrogenic as judged by the rabbit pyrogen test. The purification process is simple, inexpensive, and does not use blood components or toxic substances. The mild conditions in which the PT and FHA are purified ensure the recovery of native protein. The purified PT and FHA are detoxified in the presence of glycerol using glutaraldehyde and formaldehyde, respectively, to produce antigenic components of an acellular pertussis vaccine. The final PT and FHA toxoids are immunogenic in guinea-pigs and have been shown to be protective in the mouse intracerebral challenge test.

Bordetella pertussis↗
PubMed1991

Mapping of B-cell epitopes on the outer membrane P2 porin protein of Haemophilus influenzae by using recombinant proteins and synthetic peptides.

The P2 protein of Haemophilus influenzae type b has a porin activity and is the most abundant protein in the outer membrane. We have employed fusion protein constructs and synthetic peptides along with monoclonal antibodies to map B-cell epitopes in this protein. A linear, surface-exposed epitope was identified between residues 158 and 174. A second surface-exposed epitope was identified near the carboxy-terminal end of the protein (residues 319 to 341). Two additional B-cell epitopes were identified. One was localized between residues 28 and 55, whereas the other was located between residues 148 and 174. These epitopes were not present on the surface of intact H. influenzae cells. Thus, four distinct immunogenic and antigenic regions on the P2 protein have been identified.

Amino Acid Sequence↗
PubMed1991

Inhibition of protein-kinase C in peripheral blood mononuclear cells of patients with systemic lupus erythematosus: effect on spontaneous immunoglobulin production.

Systemic Lupus Erythematosus (SLE) is a multisystem disease characterized by an increase in the spontaneous secretion of immunoglobulin (Ig) molecules, many of which are autoreactive. We have previously shown (Biochem & Biophys. Res. Comm. (1989) 161: 1319-1326) that normal human peripheral blood mononuclear cells (PBMCs) can be stimulated to secrete large quantities of Ig upon incubation with the protein kinase-C activator 1 oleoyl-2-acetyglycerol (OAG). Specific blockage of protein kinase C with the isoquinoline sulfonyl piperazine compound (H-7) inhibited the OAG-induced Ig production. In experiments reported here, PBMC of 5 patients with active SLE produced high levels of IgG spontaneously in culture. PBMC of 6 inactive SLE patients and 7 normal control subjects produced comparable low levels of IgG spontaneously. Pokeweed mitogen (PWM) stimulation of PBMC in inactive SLE and control groups, but not active SLE patients produced markedly enhanced IgG production. The lack of response to PWM stimulation in active SLE patients is likely due to inherent maximal stimulation of active SLE B-cells. In addition, we examined the ability of H-7 to inhibit both mitogen-stimulated (normal and inactive SLE) and spontaneous (active SLE) Ig production. In other experiments, we also examined the ability of the isoquinoline sulfonamide (HA-1004), a potent inhibitor of cAMP-dependent protein kinase to regulate mitogen stimulated and spontaneous Ig production in the patient groups indicated above. H-7 significantly inhibited PWM stimulated Ig production in normal (P less than 0.0001) and inactive SLE patients, (P less than 0.040) suppressing PWM stimulated levels to spontaneous levels.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

A new Pick based computer thyroid register based on the national SAFUR requirements for local usage.

Patients with thyroid disease require lifelong surveillance to detect late onset hypothyroidism and to monitor thyroid replacement therapy. This is best achieved by a shared-care computer system. We describe the introduction to Tayside (Ninewells Hospital) of a new computerised thyroid follow-up programme based on the Patient Record System under a Pick operating system, run on a Honeywell-Bull minicomputer. This new programme has allowed the transfer of patients from the previous Aberdeen based SAFUR centralised service to a locally based unit, offering an improved, updated and cost effective method for thyroid disease surveillance. Audit, clinical and financial, may now be more efficiently achieved, allowing local needs to direct the service provision.

Continuity of Patient Care↗
PubMed1990

Simultaneous determination of Bordetella pertussis toxin and filamentous haemagglutinin concentrations by hydroxyapatite high-performance liquid chromatography.

A simple and rapid method for the simultaneous determination of Bordetella pertussis toxin (PT) and filamentous haemagglutinin (FHA) concentrations in fermentation broths has been developed. The rapid single-step analysis performed by hydroxyapatite high-performance liquid chromatography using a salt gradient with UV detection allows both the separation of PT from FHA and the measurement of their respective concentrations. The assay is highly reproducible. Over 35 lots of acellular B. pertussis vaccine production lots were examined and PT concentrations measured by high-performance liquid chromatography were found to be in good agreement with the values obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis densitometry. The chromatographic conditions have been optimized to separate the intact holotoxin from its B-oligomer subunits.

Amino Acids↗
PubMed1990

Identification of a potent synthetic HIV1 immunogen compromising gag-P24 tandem T- and B-cell epitopes.

Recent studies indicate that the gag gene products may play a crucial role in the immune response against HIV infection since clinical progression to AIDS is associated with a reduction in the level of circulating antibodies to gag p24 and antibodies raised against p17 peptide can inhibit HIV1 infection in vitro. Using conventional structure prediction algorithms for T-cell and B-cell epitopes, we have selected and chemically synthesized several gag peptides. In particular, an unconjugated HIV1-p24 peptide containing both B- and T-cell epitopes in tandem plus Freund's adjuvant induced a strong antibody response in both mice and rabbits against p24 and its precursor p55 as judged by immunoblotting. In addition, the peptide presented in the appropriate MHC context was shown to be highly stimulatory for p24 specific murine T-cell clones.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Lectinlike properties of pertussis toxin.

We have examined the lectinlike properties of pertussis toxin by binding-inhibition assays and affinity chromatography of goose erythrocyte membranes. Although pertussis toxin and wheat germ agglutinin apparently recognize similar sugar sequences on glycoproteins, the binding activities of the two lectins are not identical.

Acetylglucosamine↗
PubMed1989

Single-step purification of pertussis toxin and its subunits by heat-treated fetuin-sepharose affinity chromatography.

A general procedure for purifying biologically active pertussis toxin from Bordetella pertussis fermentation broth using affinity chromatography on heat-treated fetuin-Sepharose CL-4B is described. Diethanolamine is used as eluent in this single-step purification to prepare endotoxin-free pertussis toxin in good yield (70%) and high purity (greater than 95%). This one-step affinity chromatography procedure can be easily applied for large-scale preparation of pertussis toxin S1 subunit and its B-component. The affinity-purified S1 subunit is devoid of any of the histamine-sensitizing activity normally associated with pertussis toxin. The chromatographically purified pertussis toxin and its subunits retained their immunogenicity and could induce high levels of anti-toxin neutralizing antibodies.

Animals↗
PubMed1987

Nucleotide sequence and in vitro expression of rubella virus 24S subgenomic messenger RNA encoding the structural proteins E1, E2 and C.

The complete nucleotide sequence of the 24S subgenomic mRNA of wild-type M33 strain rubella virus has been determined. This RNA is 3,383 nucleotides in length excluding the 3'-terminal poly(A) tract. After the three multiple in-phase termination codons clustered in the 5' terminus of this RNA, there are 81 nucleotides of nontranslated nucleic acid followed by a reading frame of 2,978 nucleotides that encodes the 110 kD precursor of the structural proteins. The 3'-untranslated region is 263 nucleotides. The 110 kD polyprotein is processed to produce nucleocapsid C, the glycoproteins E2 and E1 in that order. Sites of post-translational cleavage to produce E2 and E1 were located using available N-terminal amino acid sequences. RNAs synthesized by transcription in vitro are effective messengers in the rabbit reticulocyte cell-free translation system. Post-translational processing of the structural proteins was observed in the cell-free system supplemented with microsomes from dog pancreas.

Amino Acid Sequence↗
PubMed1985

Structural analysis of a new GC-specific insertion element IS186.

A new insertion sequence, IS186, has been identified from Escherichia coli and sequenced. It contains 1336 base pairs with a terminal inverted repeat of 22 nucleotides. A long open reading frame, from an ATG codon at position 55 to a TAG termination codon at 1327, could code for a polypeptide of 424 amino acids. This element recognizes GC-rich regions as target sites for insertion.

Base Composition↗
PubMed1985

Purification of biologically active rubella virus antigens by immunoaffinity chromatography.

A general procedure for isolating biologically active rubella virus antigens (VPI, Mr = 61,000; VP2, Mr = 45,000; VP3, Mr = 36,000) by monoclonal antibody affinity chromatography is described. Complexes formed between monoclonal antibodies and rubella virus antigens were found to be stable either at low pH or in Tris buffer containing detergent and high salt, but were efficiently dissociated by 5% diethanolamine, pH 11.5, or 50 mM lithium diiodosalicylate buffer, pH 8.0. Chromatographically purified rubella viral antigens retained their antigenicity as determined by enzyme-linked immunosorbent assays. Biological studies showed that rubella structural proteins VP2 and VP3 had no hemagglutinin function while the mixture of VP1 and VP2 and VP3 directly demonstrated hemagglutination activity. These results indicate that VP1 is at least in part responsible for the hemagglutinin function of rubella virus.

Antibodies, Monoclonal↗
PubMed1981

Between-technician variation in the measurement of spirometry with air and helium.

A study using a two-period cross-over design was conducted to determine the between-technician variation in the measurement of variables derived from spirograms with subjects breathing room air and also breathing a mixture of 80% helium and 20% oxygen (He-O2). Twenty-four subjects were allocated systematically into three 2-period cross-over studies (blocks) of 8 subjects each (4 in each sequence in each block). There were 3 technicians in the study, and the subjects in each block were tested by a pair of technicians. The following variables derived from the tracings were analyzed statistically: forced expiratory volume in one second (FEV1), forced vital capacity (FVC) while breathing room air and also while breathing He-O2, maximal mid-expiratory flow rate (MMRF), FEV1/FVC %, and the per cent decrease in time between 60% VC and 40% VC for the spirograms comparing He-O2 with air (delta t). Analyses were performed to assess the presence of residual effects in the cross-over design, the between-technician difference in the administration of the tests, and in the interpretation of tracings. The results indicated that: (1) significant differences were observed between technicians in the administration of the tests to obtain FVC while the subjects breathed room air and while they breathed He-O2, (2) there were significant differences between the 3 technicians in the interpretation of MMFR, and (3) the between-technician variation in the administration of the tests with respect to delta t and with He-O2 in general were not significantly different from that of the other spirometric variables studied.

Adult↗
PubMedDate not supplied

Purification and immunological characterization of HPLC-purified pertussis toxin subunits.

Pertussis toxin (PT), an oligomeric exotoxin of Bordetella pertussis containing five dissimilar subunits, is considered to be an essential immunogen in acellular and component pertussis vaccines against whooping cough. A rapid single-step procedure for isolating PT subunits was developed using reverse-phase high-performance liquid chromatography. Recoveries of individual subunits were 75% (S1), 70% (S2), greater than 90% (S3), greater than 90% (S4), and 50% (S5), as judged by SDS-PAGE and amino acid analysis. Lyophilized subunits were solubilized in urea followed by step-wise dialysis to remove the urea. All subunits were inactive in histamine sensitization, lymphocytosis, and hemagglutination assays. However, purified S1 retained residual NAD-glycohydrolase and ADP-ribosyltransferase activity. A partially active holotoxin could be generated by mixing the five individual subunits. All subunits were immunogenic in rabbits and mice. Monospecific antisera raised in both animal species were able to neutralize the PT-mediated clustering of Chinese hamster ovary cells, but active immunization of mice with single subunits failed to protect them in the intracerebral challenge assay. These subunit preparations therefore retained neutralizing determinants, but did not contain protective epitopes.

Animals↗
PubMedDate not supplied

Synthesis and characterization of a fragment of an ice nucleation protein.

Synthetic peptides were used as models for studying the conformation of ice nucleation proteins. We chemically synthesized four peptides (16-, 24-, 32-, and 48-mer) that consisted of two to six repeats of the consensus repeating octapeptide unit of ice nucleation proteins and evaluated their conformation by circular dichroism spectroscopy. These model peptides exist predominantly as random coils in aqueous solution, but adopt alpha-helical structures in the presence of trifluoroethanol. The stability of their secondary structures was investigated by monitoring the pH and time dependence of their circular dichroism spectra. Our results indicated that the alpha-helical content of the 48-mer exhibited a significant pH dependence, while that of the 24- and 32-mer peptides did not. The 32-mer was the only peptide that transformed from the alpha-helical to a beta-sheet structure upon storage. We suggest that the overall conformation of the ice nucleation protein could be a beta-sheet.

Amino Acid Sequence↗