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Biomedical subjects

H W Chan

Publications and source records attributed to H W Chan.

At least 55 records · Page 3Linked to original sources
PubMed1990

In-111-labeled monoclonal antibody immunoscintigraphy in colorectal carcinoma: safety, sensitivity, and preliminary clinical results.

A phase I/II prospective clinical trial was performed with indium-111-labeled monoclonal antibody (MoAb) conjugate B72.3-glycyl-tyrosyl N-epsilon-diethylenetriaminepentaacetic acid (CYT-103) in 28 preoperative patients with biopsy-proved or suspected colorectal carcinomas. Immunoscintigraphy was performed 2-7 days after infusion of 4.1 mCi (152 MBq) of In-111 labeled to CYT-103 at doses of 0.5, 1.0, 2.0, and 20.0 mg. Surgical and histologic confirmation was available in all cases. Use of In-111 CYT-103 made possible detection of 75% of colorectal carcinomas at doses of 1.0 mg and higher, compared with only 20% detection at the 0.5-mg MoAb dose. Immunohistochemical staining for tumor-associated glycoprotein (TAG)-72 in resected carcinoma tissues demonstrated a positive correlation between MoAb imaging and the percentage of cells that expressed TAG-72. One patient suffered an adverse reaction after MoAb infusion. Human antimouse response to CYT-103 developed in 16% of patients.

Adenocarcinoma↗
PubMed1990

Indium-labeled anti-colorectal carcinoma monoclonal antibody accumulation in non-tumored tissue in patients with colorectal carcinoma.

Indium-111- (111In) labeled murine monoclonal antibodies ZCE 025 (against carcinoembryonic antigen) and CYT-103 MAb B72.3 (against tumor-associated glycoprotein - 72) have been used to image patients with colorectal cancers with encouraging results. The objectives of this study were to assess the frequency and causes of 111In MAb localization in tumor-free, benign tissues. Thus, scans of 75 patients who have undergone exploratory surgery following radioimmunoscintigraphy with 111In-ZCE 025 (n = 37) or 111In-CYT-103 (n = 38) were reviewed in conjunction with operative and histopathology reports. Localization in non-tumored tissues was seen in 10.8% and 13.1%, respectively, of patients receiving ZCE 025 and CYT-103. The most common sites involved were: degenerative joint disease, abdominal aneurysms, postoperative bowel adhesions, and local inflammatory changes secondary to surgery or external irradiation. Review of patients' medical history and results of concurrent diagnostic modalities is likely to lessen the false-positive rate of 111In-labeled MAb scan interpretation.

Antibodies, Monoclonal↗
PubMed1989

Soluble derivatives of the beta amyloid protein precursor of Alzheimer's disease are labeled by antisera to the beta amyloid protein.

The amyloid deposited in Alzheimer's disease (AD) is composed primarily of a 39-42 residue polypeptide (beta AP) that is derived from a larger beta amyloid protein precursor (beta APP). In previous studies, we and others identified full-length, membrane-associated forms of the beta APP and showed that these forms are processed into soluble derivatives that lack the carboxyl-terminus of the full-length forms. In this report, we demonstrate that the soluble approximately 125 and approximately 105 kDa forms of the beta APP found in human cerebrospinal fluid are specifically labeled by several different antisera to the beta AP. This finding indicates that both soluble derivatives contain all or part of the beta AP sequence, and it suggests that one or both of these forms may be the immediate precursor of the amyloid deposited in AD.

Alzheimer Disease↗
PubMed1988

Expression of K99 adhesion antigen controlled by the Escherichia coli tryptophan operon promoter.

The genetic determinant for the K99 adhesin of enterotoxigenic Escherichia coli B41 [O101:K99] has been cloned as a 7.0-kilobase BamHI-generated DNA fragment into the vector pBR322 by us and others (J. D. A. van Embden, F. K. de Graaf, L. M. Schouls, and J. S. Teppma, Infect. Immun. 29:1125-1133, 1980). Cells harboring one such construction, known as pK99-64, are capable of expressing K99 antigen on the cell surface. We replaced the natural promoter sequence for the gene encoding the K99 pilus subunit with a strong, inducible exogenous promoter, the E. coli tryptophan (trp) operon promoter, to construct the plasmid pBR-TrpK99. E. coli cells harboring pBR-TrpK99 or a similar construction in the plasmid pDR540, known as pKO-TrpK99, upon induction with 3-beta-indoleacrylic acid, produced about fourfold more K99 antigen than did cells bearing pK99-64 with the natural promoter. Expression of the pilus antigen was found to be under control of the tryptophan promoter. Plasmid instability was encountered, however, in cells bearing pKO-TrpK99 when the trp promoter was derepressed. Introduction of the aminoglycoside 3'-phosphotransferase gene of transposable element Tn5 into pKO-TrpK99 to generate pKON-TrpK99 effectively stabilized the plasmid in cells grown under identical conditions in medium containing kanamycin.

Amino Acid Sequence↗
PubMed1988

The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors.

Seven human immunodeficiency virus gag polypeptides were identified in the purified virus and in infected CD4+ lymphocytes by peptide mapping and limited amino acid sequencing of immune-purified proteins. Two gag polyproteins of 55,000 (p55) and 41,000 (p41) daltons were rapidly labeled and readily processed into the major internal gag proteins that were aligned within the gag open reading frame (ORF) as NH2-p16 (MA)-p24 (CA)-p9 (NC)-p7-COOH. The myristoylated p16 (matrix, MA) protein was processed from the myristoylated p55 gag precursor protein. The immunoreactivity of the p16 (MA) protein with region-specific gag antisera and the conservation of the N-terminal myristyl group of the p55 precursor protein in p16 (MA) confirmed its position as the N-terminal-most protein. The p9 (nucleocapsid, NC) protein was localized to residue 378 of the gag ORF, next to the C terminus of the p24/p25 (core antigen, CA) protein. The p9 protein had a repeating Cys residue containing motif which is found in the nucleic acid-binding Cys residue-containing proteins of retroviruses. The p24 (CA) protein, which was localized to residue 133 of the gag ORF, was apparently derived by C-terminal processing of an intermediate polypeptide, p25. Both the mature p24 (CA) and p16 (MA) proteins were phosphorylated at Ser residue(s). We also identified two forms of gag p41 species, one resulting from the C-terminal processing of p55 and the other originating either from N-terminal processing of p55 or from de novo synthesis.

Amino Acid Sequence↗
PubMed1988

Purification and structural characterization of the putative gag-pol protease of human immunodeficiency virus.

We have purified a 10,774-dalton protein from human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame (ORF). Radiochemical amino acid microsequencing identified 12 amino acids from the stretch of 39 N-terminal residues of this protein, beginning with a PQITLW sequence at position 69 of the pol ORF. Radiosequencing of selected tryptic peptides of the protein identified 11 additional residues (Leu-9 and Val-2) in six peptides encompassing the entire molecule of 99 residues. A protein of similar size and identical N-terminal sequence (determined through the first 39 residues) was present among the processed HIV pol gene products in Escherichia coli which expressed the entire HIV pol ORF. The C terminus of both the viral and E. coli-expressed proteins was inferred to be contiguous with the N terminus of the p64-p51 reverse transcriptase on the basis of tryptic mapping and specific immunoreactivity with an antiserum against a dodecapeptide located upstream of the reverse transcriptase. Thus, the initial processing of the pol precursor that generates the native protease is apparently preserved across phylogenetic barriers. Although the purified viral protease lacked measurable proteolytic activity, the bacterial extracts were capable of processing an HIV gag precursor protein synthesized in E. coli.

Amino Acid Sequence↗
PubMed1987

Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.

A DNA-transfection protocol has been developed that makes use of a synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). Small unilamellar liposomes containing DOTMA interact spontaneously with DNA to form lipid-DNA complexes with 100% entrapment of the DNA, DOTMA facilitates fusion of the complex with the plasma membrane of tissue culture cells, resulting in both uptake and expression of the DNA. The technique is simple, highly reproducible, and effective for both transient and stable expression of transfected DNA. Depending upon the cell line, lipofection is from 5- to greater than 100-fold more effective than either the calcium phosphate or the DEAE-dextran transfection technique.

Animals↗
PubMed1985

Solanidine is present in sera of healthy individuals and in amounts dependent on their dietary potato consumption.

Solanidine, a steroidal alkaloid, and its glycosides have been reported to have caused poisoning in man and animals. These alkaloids are normally present in small amounts in potatoes. Measurement of solanidine in body fluid would be expected to establish the real incidence of acute toxicity and help to resolve the question of any chronic toxicity including teratogenicity. We report the detection of solanidine in the serum of 57 normal healthy volunteer subjects in whom it measured 4.0-56.3 nmol/l (1.6-22.5 ng/ml) before the midday meal. There was a significant correlation between serum solanidine concentration and normal dietary intake of potato by the individual concerned. When two subjects abstained from potato and its products serum solanidine fell markedly and became minimal after the second week onwards.

Adolescent↗
PubMed1983

Determination of solanidine in human plasma by radioimmunoassay.

A radioimmunoassay for solanidine, the major hydrolysis product of potato glycoalkaloids, has been established and validated for application to human plasma. All 34 samples of human plasma tested contained solanidine, with a mean of 1.27 +/- 0.97 ng/ml (3.18 +/- 2.43 nmol/litre).

Adult↗
PubMed1983

Class I defective herpes simplex virus DNA as a molecular cloning vehicle in eucaryotic cells.

Defective herpes simplex virus type 1 genomes are composed of head-to-tail tandem repeats of small regions of the nondefective genome. Monomeric repeat units of class I defective herpes simplex virus genomes were cloned into bacterial plasmids. The repeat units functioned as replicons since both viral and convalently linked bacterial plasmid DNA replicated (with the help of DNA from nondefective virus) when transfected into rabbit skin cells. Recombinant plasmids were packaged into virions and were propagated from culture to culture by infection with progeny virus. Replication was evidently by a rolling circle mechanism since plasmid DNA was present in a high-molecular-weight form in transfected cells. Circular recombinant plasmid DNA replicated with a high degree of fidelity. In contrast, linear plasmid DNA underwent extensive deletions of both viral and bacterial sequences when transfected into rabbit skin cells. Derivative plasmids, a fraction of the size of the parental plasmid, were rescued by transforming Escherichia coli with DNA from the transfected rabbit skin cells. These plasmids functioned as shuttle vectors since they replicated faithfully in both eucaryotic and procaryotic cells.

Animals↗
PubMed1983

Identification of nonhistone chromosomal proteins binding specifically to cloned alpha-fetoprotein DNA sequences.

A sequential nitrocellulose filter binding assay has been used to quantitate specific binding by nonhistone proteins of a cloned DNA fragment coding for the 3' end of the mouse alpha-fetoprotein gene. Nonhistone proteins from fetal mouse liver, from adult liver, and from kidney, all bind this fragment specifically; there is no significant difference in binding by proteins isolated from these three tissues. The proteins responsible for specific binding have been isolated by sucrose density gradient ultracentrifugation of DNA-protein complexes and characterized by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and electron microscopy. The majority of the specific binding proteins from adult liver consists of two polypeptides of molecular weights 53 500 and 51 000; three or four minor bands in the molecular weight range 43 000-48 000 are also observed. Similar specific binding proteins exist among the fetal liver nonhistone proteins. The native binding proteins as visualized by electron microscopy are very large and appear to bind specifically to a site near the centre of the alpha-fetoprotein DNA fragment, as well as nonspecifically to the ends of all DNA molecules.

Animals↗
PubMed1983

Enzyme-linked immunosorbent assay of ochratoxin A in barley.

A noncompetitive, double antibody enzyme-linked immunosorbent assay for ochratoxin A using microtitration plates has been developed and applied to samples of barley. The anti-ochratoxin A antiserum, which is used at high dilution, does not cross-react significantly with ochratoxin B or ochratoxin a. Assay sensitivity for determination of the toxin in barley samples is 60 ng/kg. Minimal sample preparation is required before assay.

Enzyme-Linked Immunosorbent Assay↗
PubMed1982

Cloning and characterization of an envelope-specific probe from xenotropic murine leukemia proviral DNA.

An 8.9-kilobase EcoRI restriction fragment was cloned from mink cells chronically infected with NFS-Th-1 xenotropic murine leukemia virus by using a lambda phage host vector system. After its transfer into pBR322, the EcoRI DNA insert was characterized and found to contain 6.7 kilobases of proviral DNA sequences and 2.2 kilobases of mink cellular DNA flanking the 5' end of the viral genome. A 500-base pair fragment which was located at the 3' terminus of the cloned DNA insert and which mapped to the env region of xenotropic proviral DNA was subcloned into pBR322. This xenotropic envelope proviral DNA segment did not hybridize to ecotropic murine leukemia proviruses but did anneal to representative alpha and beta xenotropic and seven different mink cell focus-inducing proviral DNAs. The cloned xenotropic envelope-specific probe was also used in blot hybridization experiments to analyze the arrangement of related sequences in preparations of different mouse liver DNAs.

Animals↗
PubMed1982

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA.

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA.

AKR murine leukemia virus↗
PubMed1982

Two defective DNAs of human polyomavirus JC adapted to growth in human embryonic kidney cells.

Human polyomavirus JC (JCV) adapted to growth in human embryonic kidney (HEK) cells contains two or more species of shorter-length viral DNA even after two cycles of plaque purification in HEK cells. We have molecularly cloned JCV DNA from one plaque isolate and determined the physical map of its DNA. Using gel electrophoresis and electron microscopy, we found that the cloned DNA consisted of two classes of JCV DNA. One class of DNA had a deletion of 30% (between 0.7 and 1.0 map units from the EcoRI site) and an insertion of 1% at the same site in the late region. The other had a deletion of 35% (0.18 to 0.53 map units) and an insertion of 12% at the same site in the early region of the JCV genome. Both DNAs had added sequences near the origin of DNA replication. From their structure, the two DNAs appear to complement each other. These results indicate that the HEK-adapted JCV may be replicating by complementation between two defective mutants.

Cell Line↗
PubMed1982

Internal organization of endogenous proviral DNAs of xenotropic murine leukemia viruses.

The internal organization of endogenous xenotropic murine leukemia virus proviruses was determined in a series of blot hybridization experiments in which DNA from several different inbred mouse strains, digested with restriction enzymes known to cleave xenotropic proviral DNAs at least twice, was annealed to generalized murine leukemia virus or xenotropic env-specific DNA probes. Comigrating bands of variable intensity which hybridized to the xenotropic env probe were identified in all inbred mouse DNA preparations. At least seven classes of endogenous xenotropic proviral DNA with respect to SacI cleavage maps were detected in mouse DNA. Two of the seven classes were indistinguishable from proviruses associated with known infectious xenotropic murine leukemia viruses. These results are consistent with the existence of related but organizationally distinct families of endogenous xenotropic proviral DNA that are present in different relative abundances in mouse genomic DNA.

Animals↗
PubMed1982

Identification of ecotropic proviral sequences in high- and low-ecotropic-virus-producing mouse strains.

The arrangement of endogenous ecotropic retroviruses in selected high- and low-ecotropic-virus-producing mouse strains was examined by Southern blot hybridization analysis, using an ecotropic retrovirus-specific DNA probe. High-ecotropic-virus-producing mouse strains of the AKR family displayed heterogeneity with respect to the number of copies and the sites of insertion of endogenous ecotropic specific DNA. This diversity was seen even among individuals of the same AKR subline. Contrastingly, individuals within the same low-ecotropic-retrovirus-producing mouse strain showed no evidence of variability in their endogenous ecotropic proviral sequences. These results favored the hypothesis that germ line proviral reinsertion was responsible for the proviral sequence heterogeneity observed in high-ecotropic-virus-producing mouse strains.

AKR murine leukemia virus↗