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Biomedical subjects

K B Choo

Publications and source records attributed to K B Choo.

At least 55 records · Page 3Linked to original sources
PubMed1991

Immunoblot analysis of components of Penicillium notatum recognized by human IgE antibodies.

Components of the crude extract of Penicillium notatum recognized by human IgE antibodies (Abs) were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The allergenic components were identified with sera from 19 allergic patients and 20 blood donors. The allergen-Ab complexes were visualized by 125I-labeled monoclonal antihuman IgE and autoradiography. A total of 11 allergenic components, ranging in molecular weights (MWs) from 94,000 to 20,000 daltons, were identified. Heterogeneity in the IgE-binding patterns of the serum samples tested was also observed. However, the major allergen appears to be the component with an MW of about 68,000 daltons that was recognized by IgE Abs in 56% of the 39 sera analyzed. Furthermore, the component with an MW of about 64,000 daltons that was recognized by IgE Abs in 46% of the sera analyzed was also considered as an important allergen. Results obtained from this study will be useful in additional characterization of allergens of P. notatum and related fungal species.

Allergens↗
PubMed1991

The 40-kilodalton allergen of Candida albicans is an alcohol dehydrogenase: molecular cloning and immunological analysis using monoclonal antibodies.

To characterize the 40-kilodalton (kD) major allergen of Candida albicans (C. albicans), six monoclonal antibodies (MoAbs) against this allergen were generated. In SDS-polyacrylamide gel electrophoresis and immunoblot analysis, these MoAbs showed four different reaction patterns to antigens of six different Candida species. With the exception of one MoAb, other MoAbs were resistant to periodate treatment indicating non-carbohydrate epitopes were probably being recognized by these MoAbs. These MoAbs were used in the molecular cloning and immunological analysis of the gene coding for the 40-kD allergen. Nucleotide sequence determination of the two lambda gt11 cDNA clones obtained showed that the 40-kD allergen is an alcohol dehydrogenase (ADH) which shares a 70% amino acid sequence homology with the ADH isozyme I of Saccharomyces cerevisiae. This finding was confirmed by positive immunological response of the lysates of the clones obtained and a preparation of ADH of Saccharomyces cerevisiae to various MoAbs and to IgE antibodies in sera of allergic patients.

Alcohol Dehydrogenase↗
PubMed1991

Characterization of a monoclonal antibody (RJ5) against the immunodominant 41-kD antigen of Candida albicans.

A 41-kD component of Candida albicans was identified to be the major antigen radioimmunoprecipitated by antibodies with increased titers in the sera of patients with invasive candidiasis. A mouse monoclonal antibody (RJ5) was generated which, by immunoblotting, showed positive reactivity to the immunoprecipitated 41-kD component. By two-dimensional gel electrophoresis and immunoblotting, MoAb RJ5 was shown to react with different isoforms of the 41-kD component with pI values from 6.1 to 6.9. Furthermore, MoAb RJ5 showed positive reactivity to cytoplasmic antigens of C. albicans by frozen section and immunoperoxidase staining. By SDS-polyacrylamide gel electrophoresis and immunoblotting, MoAb RJ5 showed no cross-reactivity to antigens of Candida tropicalis and Candida parapsilosis. The epitope of the 41-kD molecule recognized by MoAb RJ5 was susceptible to treatment of proteinase K at concentrations of greater than or equal to 5 micrograms/ml, and was relatively resistant to periodate oxidation with concentration of NaIO4 up to 20 mM. This MoAb may be useful in the purification and characterization of the immunodominant 41-kD antigen of C. albicans, and as a probe in the detection of Candida antigens in the sera of patients with invasive candidiasis.

Antibodies↗
PubMed1991

Detection of beta-globin gene mutations by polymerase chain reaction.

The polymerase chain reaction and oligonucleotide probe hybridization technique were applied to the detection of two common mutations of the beta-globin gene found in Chinese, namely the 4-base pair deletion at the 41-42 codons and the C to T substitution at nucleotide 654 of IVS-2. The accuracy of the method was established using beta-thalassemia cases with known mutations or haplotypes of the restriction fragment length polymorphism (RFLP). A further 11 cases of thalassemia intermediate and thalassemia minor were then analysed with the same approach. Our results showed that 5 of the 11 cases carried the TCTT-deletion at codons 41-42. Our method is economical both in terms of materials and time needed and in an alternative to the use of the molecular RFLP approach in the prenatal diagnosis of beta-thalassemia.

Base Sequence↗
PubMed1990

An improved scheme for the identification of antigens recognized by specific antibodies in two-dimensional gel electrophoresis and immunoblotting.

This paper describes an improved scheme for the identification of antigens in crude extracts recognized by specific antibodies when analyzed by a combination of two-dimensional gel electrophoresis and immunoblotting. First, protein components in gels are electrophoretically transferred to a polyvinylidene difluoride membrane which does not shrink or change dimensions in organic solvents. The efficiency of transfer and the localization of sample proteins on the membrane are checked and recorded by staining the blotting membrane with Fast Green FCF and recording the profile on a transparency. After blocking and the immunoassay, the results are recorded by photography. The sites of immune reaction are marked and the same membrane is restained briefly with Coomassie Brilliant Blue R-250 for the protein profile. Thus antigens in complex mixtures, recognized by antibodies of interest, can easily be identified from the restained membrane. If the whole protein profile is not well demonstrated, when used in combination with the profile recorded on the transparency, spots appearing on the restained membrane can still be used as useful landmarks in the final unequivocal antigenic identification. This improved scheme circumvents problems arising from membrane shrinkage and difficulties in accurately matching immunoreactive spots by conventional procedures and thus provides an accurate, simple and fast approach in the identification of antigens after immunoblotting.

Antibodies, Monoclonal↗
PubMed1990

Analysis of the unoccupied site of an integrated human papillomavirus 16 sequence in a cervical carcinoma.

We have previously cloned and analyzed the structure of a type 16 human papillomavirus (HPV16) integration in a primary cervical carcinoma tissue, M50 (Choo et al., J. Virol. 62, 1659-1666, 1988). We found that specific nucleotide sequences within the HPV16 genome influenced the genomic organization of the integrated viral genome. Using the viral-cellular junctions of the M50 DNA as probes, we have now cloned the unoccupied site from a human genomic library. Mapping analysis showed that a deletion of about 1.1 kilobase pairs (kb) had occurred at the integration site of M50. Sequencing of the integration junctions of the unoccupied site and comparison with the viral sequence has revealed short regions of sequence homology between the viral and the cellular genomes at both junctions. Our results are consistent with a mechanism of integration of the HPV16 sequences in the M50 carcinoma involving illegitimate recombination events using short patches of homologous sequences between the two heterologous genomes for anchorage and as guides for crossover. Preferred topoisomerase I cleavage sites and alternating purine and pyrimidine bases, which favor the formation of Z-DNA, could also be identified at the integration regions, supporting a proposed role for the topoisomerase I enzyme in the illegitimate recombination in the viral integration process.

Attachment Sites, Microbiological↗
PubMed1990

Detection of human papillomavirus DNA in human cervical lesions by tissue in situ nucleic acid hybridization.

Cervical cancer is one of the most common female cancers in Taiwan. Certain types of human papillomavirus (HPV) are frequently detected in the epithelial precancerous and cancerous lesions of the cervix. By the use of tissue in situ hybridization, we investigated the relationship of various types of HPV (group I, HPV-6 & 11, group II, HPV-16 & 18, group III, HPV-31, 33 & 35) with cervical condyloma, carcinoma as well as precancerous lesions. Group I HPV DNAs were mainly found in cervical condylomatous lesions (2/2) of the cervix and cervical intraepithelial neoplasia I (CIN I) (2/4), but were only occasionally found in CIN II (1/4), CIN III (1/9) or non-keratinized squamous cell carcinoma (1/15). HPV DNAs of groups II and III were mainly detected in lesions of CIN III (5/9) and invasive squamous cell carcinoma (large cell, keratinized type: 4/7; large cell, non-keratinized type: 11/15). HPV DNA sequences were invariably detectable only in the cell nuclei of condyloma or dysplastic epithelium or invasive carcinoma. However, they could not only be detected in the upper layer dysplastic cells and koilocytes but also in the well and poorly differentiated cervical cancer cells. The distribution of HPV DNA positive cells in the carcinomas fell into four different patterns: (1) upper zone and non-invasive regions of the carcinoma (11/22, 50%), (2) basal zone and invasive regions (2/22, 9%), (3) randomly scattered (7/22, 32%), and (4) extensively distributed over the whole tumor lesions (2/22, 9%). Thus, our results are consistent with a strong correlation between the presence of HPV-16, 18, 31, 33 and 35 and malignant conversion of cervical epithelial cells.

Condylomata Acuminata↗
PubMed1990

Rapid verification of lambda-cDNA clones using mixed-base oligonucleotide as screening probe and sequencing primer.

A rapid procedure has been developed for the isolation and verification of cDNA clones isolated from a cDNA library based on lambda vectors. Using information of the partial amino acid sequence of a protein, synthetic mixed-base oligonucleotides are first employed as a screening probe using the plaque hybridization procedure. The cDNA inserts of the clones obtained are then directly amplified by polymerase chain reaction (PCR) using primers flanking the cloning site of the vector. Besides being used for cloning into a plasmid vector, the amplified DNA's are also subjected to nucleotide sequence analysis using the same mixed-base oligonucleotides as sequencing primers. This approach allows sequencing through the region of the known amino acid sequence for direct verification of the authenticity of the clones obtained. This procedure has successfully been used for cloning and partial characterization of the gene coding for a platelet aggregation inhibitor.

Amino Acid Sequence↗
PubMed1989

Analysis of the structure and expression of the c-myc oncogene in cervical tumor and in cervical tumor-derived cell lines.

The structure of the c-myc oncogene in 17 cervical tumors and patient-matched nontumor tissues from Chinese patients residing in Taiwan was analysed. In contrast to recent reports on Mexican patients, none of the samples showed rearrangements and sequence amplification in the c-myc gene. The discrepancy may be explained by different carcinogenesis mechanisms being in operation in different geographic regions. Although no structural alterations in the c-myc gene were found in seven cervical carcinoma cell lines analysed, Northern blot analysis indicated different levels of c-myc gene expression which may be related to the presence of human papillomavirus (HPV) sequence in the cell and suggests a possible c-myc-hpv interaction in some stages of the transformation process.

Blotting, Northern↗
PubMed1989

High prevalence of human papillomavirus infection and possible association with betel quid chewing and smoking in oral epidermoid carcinomas in Taiwan.

Seventeen oral epidermoid carcinomas, three oral papillomas, and 17 normal gingival tissues were tested for the presence of human papillomavirus (HPV) types 6, 11, 16, and 18 sequences by Southern blot hybridization. Episomal HPV-16 sequences in various amounts were detected in 76.4% of the oral carcinomas and in all three cases of papilloma. However, only one of the 17 normal tissues was HPV positive with an unknown type. None of the samples contained HPV-6, -11, or -18 sequences. Examination of the habits of the patients showed that 59% of the patients were betel quid chewers and 82% were smokers. Thus, the concurrent incidence of HPV infection and betal quid chewing and/or smoking habits in oral carcinoma patients observed in Taiwan is consistent with the view that both viral and chemical factors may be involved in the process of carcinogenesis.

Areca↗
PubMed1989

Allergenic components of Candida albicans identified by immunoblot analysis.

Allergenic components of Candida albicans fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes were identified using sera from 30 asthmatic patients who showed positive skin test and RAST (radio-allergosorbent test) to C. albicans. The IgE-binding yeast components in the complex antigen preparation were then detected by reaction with enzyme-labelled anti-human IgE antibodies. They were confirmed by Coomassie blue R-250 staining of the membrane to visualize all protein bands after reaction with the enzyme substrate. The IgE-binding patterns of the sera tested were heterogeneous, displaying a total of 16 identifiable components with molecular weights ranging from 20 to 94 kD. A 40 kD component showed the highest IgE-binding frequency, being recognized by 23 (77%) of the 30 sera examined. The other 15 allergenic components identified were recognized by less than 25% of the sera tested. Only two of the 30 serum samples contained IgE antibodies reactive with seven to eight allergenic components. Ten of the 30 sera reacted with only one allergenic component, and the remaining serum samples recognized two to five of the 16 identified allergens. Results described in this study are applicable to allergen standardization work and provide a basis for further study on the role of C. albicans in clinical allergy.

Antigens, Fungal↗
PubMed1989

Presence of catenated human papillomavirus type 16 episomes in a cervical carcinoma cell line.

Human papillomavirus (HPV) is frequently associated with cervical carcinoma and derived cell lines. In primary tissues of the carcinoma, the viral genome may be present in episomal or integrated configuration. In cell lines, however, only integrated HPV sequences have been reported. In this article, we describe the presence of episomal type 16 HPV (HPV16), demonstrated by electron microscopy and two-dimensional agarose gel electrophoresis, in a cervical carcinoma cell line, CC7T/VGH, established in 1980 in Taiwan. In CC7T/VGH, the HPV16 sequences are transcriptionally active, and at least three major HPV16 RNA species were detected in Northern blots. Results from restriction enzyme and S1 nuclease analysis suggest a composition of oligomeric HPV16 molecules in dimeric repeats. In addition, the HPV16 oligomers exist as catenated molecules of interlocking rings instead of concatemers. A monomeric copy of the HPV16 episome was cloned from a Hirt supernatant of CC7T/VGH by using a plasmid vector. Mapping and partial sequencing studies revealed an internal deletion of 163 base pairs within the L1 open reading frame. However, insertion of an A.C nucleotide pair at the deletion junction restored the otherwise frame-shifted L1 open reading frame. Two base transitions were also found within the E7 and the E1 open reading frames. Our findings suggest the need for closer examination for HPV episomal catenation in other cervical carcinoma cell lines as well as in primary carcinoma tissues of the uterine cervix and the anogenital tract. With CC7T/VGH, a way is now available for studies of many important aspects of the biology of HPV such as replication and gene expression of the extrachromosomal viral genome.

Base Sequence↗
PubMed1989

Arrangements of alpha-globin gene cluster in Taiwan.

In a gene mapping study on 217 newborn babies in Taiwan with alpha- and zeta-globin probes, we have observed 4 cases (1.84%) of alpha-thalassemia-2 heterozygotes (zeta zeta-alpha/zeta zeta alpha alpha) without increased levels of hemoglobin (Hb) Bart's in the cord blood. Eleven subjects (5.07%) were found to have the South East Asian alpha-thalassemia-1 haplotype (zeta zeta--SEA/zeta zeta alpha alpha) with increased Hb Bart's levels ranging from 2.2 to 9%. One case, with Hb Bart's level of 14% in the cord blood, was found to have the genotype of zeta zeta--SEA/zeta zeta alpha alpha T (0.46%). Four heterozygotes (1.84%) were found with the triple alpha gene anti-rightward arrangement (zeta zeta alpha alpha alpha 3.7/zeta zeta alpha alpha). Twenty-one heterozygotes (9.68%) were found to have the triple zeta-globin gene arrangement (zeta zeta zeta alpha alpha/zeta zeta alpha alpha). A new triple zeta-globin gene variant with a BamHI polymorphism was also observed in this study.

Asian People↗
PubMed1989

Production of transgenic mice carrying the human hepatitis B virus or the human papillomavirus DNA sequences in Taiwan: analysis of physical structure and hereditary mode.

In order to produce suitable animal model systems for the analysis of possible synergistic interaction between hepatitis B virus (HBV) or the human papillomavirus (HPV) with other viral or chemical co-factors, we have produced transgenic mice carrying HBV, HPV16 or HPV18. Transfer of 3343 micro-injected one-cell (C57/BL6 X CBA) F1 embryos into pseudo-pregnant ICR recipients produced 181 pups. Seventeen of 151 of the pups analysed were found to be transgenics. Southern blot and slot blot analysis showed that the copy number of the transgenome in the transgenic mice ranged from 1-50 copies per cell. All the transgenomes analysed appeared to be in tandem repeats without detectable sequence rearrangements. The transgenome sequences were also stable on transmission to the F3 generation. On breeding, six of the ten transgenic mice analysed showed the Mendelian mode of inheritance with integration at single or multiple loci. Of the remaining four mice, three were mosaics and one was sterile. Analysis of the gene expression of the transgenic mice is currently in progress.

Animals↗
PubMed1989

Infectivity in asymptomatic hepatitis B carriers: its correlation with HBeAg/anti-HBe status.

Sera collected from 190 asymptomatic HBsAg carriers with varying status of HBe markers were tested for HBV DNA using the slot-blot hybridization method and the results were compared with serum alanine aminotransferase (ALT) levels. No significant difference was observed in the positive rate of serum HBV DNA between patients showing either HBeAg (+)/normal ALT or HBeAg (+)/high ALT. On the other hand, in cases with positive anti-HBe or negative for both HBeAg and anti-HBe, statistically significant differences could be shown in HBV DNA positivity between normal and high ALT subjects. In the group of 100 patients possessing anti-HBe antibody, 50% of the cases with high ALT levels were positive for serum HBV DNA, whereas all of the cases with normal ALT levels were negative for serum HBV DNA (p less than 0.0002). In 21 patients showing HBeAg (-)/anti-HBe(-), HBV DNA could be detected in the serum of 60% of the cases with high ALT levels, but in none of the cases with normal ALT levels (p less than 0.05). Our results suggest that HBeAg alone is a reliable marker in the prediction of infectivity in asymptomatic carriers. In cases showing anti-HBe (+), loss of infectivity could be ascertained if the patient had normal serum ALT, but, continuous viral replication should be suspected in cases with high serum ALT values.

Adolescent↗
PubMed1988

Differential identification of Candida species and other yeasts by analysis of [35S]methionine-labeled polypeptide profiles.

This paper describes a scheme for differential identification of Candida species and other yeasts based on autoradiographic analysis of protein profiles of [35S]methionine-labeled cellular proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using ATCC strains as references, protein profile analysis showed that different Candida and other yeast species produced distinctively different patterns. Good agreement in results obtained with this approach and with other conventional systems was observed. Being accurate and reproducible, this approach provides a basis for the development of an alternative method for the identification of yeasts isolated from clinical specimens.

Candida↗
PubMed1988

Sequence duplication and internal deletion in the integrated human papillomavirus type 16 genome cloned from a cervical carcinoma.

Integrated human papillomavirus type 16 (HPV16) sequences were cloned from a cervical carcinoma and analyzed by restriction mapping and nucleotide sequencing. The viral integration sites were mapped within the E1 and E2 open reading frames (ORFs). The E4 and E5 ORFs were entirely deleted. An internal deletion of 376 base pairs (bp) was found disrupting the L1 and L2 ORFs. Sequencing analysis showed that an AGATGT/ACATCT inverted repeat marked the deletion junction with two flanking direct repeats 14 and 8 bp in length. A 1,330-bp sequence duplication containing the long control region (LCR) and the E6 and E7 ORFs was also found. The duplication junction was formed by two 24-bp direct repeats with 79% (19 of 24) homology located within the LCR and the E2 ORF of the prototype viral genome, respectively. This observation leads us to propose that the initial viral integration involved an HPV16 dimer in which the direct repeats in tandem units recombined, resulting in reiteration of only a portion of the original duplication. A guanosine insertion between nucleotides 1137 and 1138 created a continuous E1 ORF which was previously shown to be disrupted. Results from this study indicate that sequence reiteration and internal deletion in the integrated, and possibly in the episomal, HPV16 genome are influenced by specific nucleotide sequences in the viral genome. Moreover, reiteration of the LCR/E6/E7 sequences further supports the hypothesis that the E6/E7 ORFs may code for oncogenic proteins and that regulatory signals in the LCR may play a role in cellular transformation.

Base Sequence↗