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Biomedical subjects

K B Choo

Publications and source records attributed to K B Choo.

At least 19 recordsLinked to original sources
PubMed2001

In silico mining of EST databases for novel pre-implantation embryo-specific zinc finger protein genes.

Progress in the understanding of early mammalian embryo development has been severely hampered by scarcity of study materials. To circumvent such a constraint, we have developed a strategy that involves a combination of in silico mining of new genes from expressed sequence tags (EST) databases and rapid determination of expression profiles of the dbEST-derived genes using a PCR-based assay and a panel of cDNA libraries derived from different developmental stages and somatic tissues. We demonstrate that in a random sample of 49 independent dbEST-derived zinc finger protein genes mined from a mouse embryonic 2-cell cDNA library, more than three-quarters of these genes are novel. Examination of characteristics of the human orthologues derived from these mouse genes reveals that many of them are associated with human malignancies. Expression studies have further led to the identification of three novel genes that are exclusively expressed in mouse embryos before or up to the 8-cell stage. Two of the genes, designated 2czf45 and 2czf48 (2czf for 2-cell zinc finger), are zinc finger protein genes coding for a RBCC protein with a RFP domain and a protein with three C2H2 fingers, respectively. The third gene, designated 2cpoz56, codes for a protein with a POZ domain that is often associated with zinc finger proteins. These three genes are candidate genes for regulatory or other functions in early embryogenesis. The strategy described in this report should generally be applicable to rapid and large-scale mining of other classes of rare genes involved in other biological and pathological processes. Mol. Reprod. Dev. 59:249-255, 2001.

Animals↗
PubMed2001

Inhibition of thymidylate synthase activity by antisense oligodeoxynucleotide and possible role in thymineless treatment.

Thymidylate synthase (TS) is an important target for chemotherapeutic treatment of cancer. However, efficacy of TS-targeted anticancer drugs is limited by the development of drug resistance as a result of TS gene amplification. In this work, a phosphorothioated antisense oligonucleotide (ODN), designated ATS-2, was used to suppress cellular synthesis of TS. ATS-2 at 0.2 microM concentration was mixed with lipofectin in a charge ratio of 1:1 and was used to treat the human embryonic kidney (HEK) cell line. A reduction of TS mRNA and protein was achieved. Furthermore, a dose-dependent reduction of cumulative viable cells of up to 98% was observed. Flow cytometer analysis of cell cycle progression indicates that ATS-2-treated cells were arrested and went into apoptosis at the S phase, possibly because of thymidine shortage, suggesting that ATS-2 is specifically effective for dividing cells. When used in combination with the anticancer drug FdUrd, ATS-2 exerted a additive inhibitory effect on cellular proliferation. To elucidate the possible role of cellular thymidine kinase (TdR kinase) in ATS-2 treatment, a second cell line, HeLa, was used. Both HEK and HeLa have similar rates of cell division and ODN uptake. In contrast to HEK, which was shown to have very low levels of TdR kinase activity in [(3)H]thymidine incorporation experiments, [(3)H]thymidine incorporation in HeLa was 15-fold greater than that of HEK. We found that HeLa cells were sensitive to FdUrd but were rather resistant to ATS-2. On the contrary, HEK cells were sensitive to ATS-2 but insensitive to FdUrd. Effects of ATS-2 and FdUrd are, therefore, complementary in thymineless treatment too.

Antimetabolites, Antineoplastic↗
PubMed2000

Identification of genes expressed in the epithelium of porcine oviduct containing early embryos at various stages of development.

As a first step toward elucidation of the action of factors secreted by the epithelium of oviduct, differential display reverse transcription-polymerase chain reaction (DDRT-PCR) was used in this study to identify transcripts of such oviductal factors in gilts carrying various stages of early embryo development post hormone-induced ovulation. A total of 13 differentially expressed transcripts were identified between 50 and 120 hr post-hCG injection (between 1- and 8-cell embryonic stages). Twelve of these transcripts were found to be initially expressed at 96 hr post-hCG injection (at 4-cell embryonic stage) and beyond. Three of such genes were shown by sequence analysis to be the porcine transforming growth factor-alpha, the porcine transforming growth factor-beta-binding protein II and a porcine astral natriuretic factor receptor-like transcript. Only one differentially expressed gene was detected between 50-60 and 85 hr post-hCG injection, and this gene turned out to be the porcine follicle-stimulating hormone receptor. The remaining eight transcripts detected by DDRT-PCR were novel. Moreover, most of these newly expressed genes were found to be turned on at a time coincidental with that of the 4-cell block of porcine embryos cultured in vitro. Our results demonstrate that DDRT-PCR is a feasible approach for rapid identification of genes that are differentially expressed in oviductal epithelium. Some of the genes thus identified may be important for unhindered development of embryos in the oviduct.

Animals↗
PubMed2000

Analysis of relative binding affinity of E7-pRB of human papillomavirus 16 clinical variants using the yeast two-hybrid system.

A number of genotypes of the human papillomaviruses (HPV) are associated with malignancies of the uterine cervix. Sequencing work has revealed the existence of intratype HPV variants with minor differences in the nucleotide sequence. More recent data suggest the possibility that some of the variants may have different modes of clinical manifestation. In this study, sequences of the E6 and E7 oncogenes of 17 HPV16 isolates derived from PAP smear samples of Taiwanese patients were analyzed. A number of E6 and E7 novel variants were found. Particularly, a prevalent (64.7%) E6 polymorphic site A442C with an E113D amino acid substitution seems specific to Taiwanese patients. In E7, two novel but silent polymorphic sites G663A (41.2%) and T846C (88.2%) were also prevalent in the samples analyzed. The yeast two-hybrid system was adopted for rapid assessment of relative E7-pRb binding affinity in the variants. The relative binding affinities of the E7 proteins of different HPV types to pRB were in close agreement with previous biochemical data. A T663G/C24W polymorphic change in E7 correlated with a decrease in E7-pRb relative binding affinity the significance of which remains to be clarified. This semi-quantitative biochemical and genetic approach may be useful as a first step in the development of clinical protocols for the screening and identification of important HPV variants for clinical interpretation and for further functional analysis by transfection or other bioassays.

Female↗
PubMed1999

Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene.

A sensitive technique for the sexing of bovine embryos was developed using polymerase chain reaction (PCR) amplification of the bovine amelogenin (bAML) gene on the X- and Y-chromosomes of Holstein dairy cattle. Cloning and DNA sequencing showed a 45.1% homology between the fifth intron of the bAML-X and bAML-Y gene with multiple deletions. A pair of sex-specific primers was designed to allow amplification of a single fragment of 467-bp from the X-chromosome of female cattle and two fragments of 467-bp and 341-bp from the X- and Y-chromosomes of male cattle. The primers were successfully applied to bovine sexing from single blastomeres isolated from day-6 to day-7 cow embryos by direct cell lysis and PCR. Our protocol of embryo sexing should be applicable to the diagnosis of defective genes in vitro in human embryos and in other domestic or recreational animals.

Amelogenin↗
PubMed1999

A point mutation in the albumin gene in a Chinese patient with familial dysalbuminemic hyperthyroxinemia.

Familial dysalbuminemic hyperthyroxinemia (FDH) is an autosomal dominant disorder characterized by euthyroid hyperthyroxinemia. However, FDH has not been reported in Chinese or African patients. Here, we report the first case of FDH in a Chinese patient. A 69-year-old Chinese man was found to have increased serum total T(4) concentrations (198-242nmol/l; normal range 58-148nmol/l) and free T(4) concentrations (>58pmol/l; T(4) analog method, normal range 9-28pmol/l). Serum total T(3) and TSH concentrations were normal. The patient was misdiagnosed as hyperthyroid and was later suspected to have a TSH-producing tumor by the finding of a pituitary microadenoma, which was eventually proven to be a non-functional pituitary 'incidentaloma'. Electrophoretic analysis of the patient's serum proteins demonstrated enhanced albumin binding of [(125)I]T(4). Serum free T(4) concentrations were normal (16-19pmol/l, normal range 9-26pmol/l) when a two-step method was used. Direct sequencing of the albumin gene showed a guanine to adenosine transition in the second nucleotide of codon 218, resulting in a substitution of histidine (CAC) for the normal arginine (CGC) in one of the two alleles in the patient. The point mutation was further confirmed by HphI digestion of exon 7 of the albumin gene. The patient's son was not affected. Our studies demonstrated that the point mutation of the albumin gene in a Chinese patient with FDH was similar to that found in western white families, but differed from that in a Japanese family in whom a guanine to cytosine transition at the same position was found.

Aged↗
PubMed1999

Semiautomated typing of human papillomaviruses by restriction fragment length polymorphism analysis of fluorescence-labeled PCR fragments.

A simplified version of a PCR-based reductional restriction fragment length polymorphism (rRFLP) approach for typing of human papillomaviruses (HPVs) is described previously [Wang et al., 1997]. It is achieved by the use of a biotin-labeled primer in PCR which, on restriction digestion and staining, is associated with only a single restriction fragment. In this report, we describe a further development of the rRFLP approach with the use of a fluorescence-labeled primer in PCR and fragment detection by laser scanning in an automatic sequencer. HPV typing is achieved by computer-assisted matching of the fluorescence-labeled rRFLP patterns with a database of rRFLP patterns of all known anogenital HPV types. On analysis of the typing of 133 HPV-positive cases using this procedure, 20 different HPV types were detected in exfoliated cervical cells in PAP smear samples derived from Taiwanese women. The results indicate the existence of a heterogeneous population of HPV types in Taiwan. Although most cases were associated with the more common HPV types, a significant fraction (about 20%) of the HPV types detected was related to the less common genotypes, which are often not included in commercial kits available for HPV typing. The results indicate the importance of covering as many HPV types as possible in clinical HPV genotyping protocols.

Cervix Uteri↗
PubMed1998

Intrahepatic vein fetal blood sampling: current role in prenatal diagnosis.

OBJECTIVE: To identify the site of fetal blood sampling (FBS) with lesser complications; and also analyses the reasons for targetting the intrahepatic vein (IHV) for FBS. METHODS: Fetal blood sampling (FBS) performed on 382 women over a period of 7 years at the National University Hospital, Singapore was analysed. FBS was performed from 13 weeks of gestational age onwards. In 76.4% (292 of 382) the intrahepatic part of the umbilical vein (IHV) was targetted; in 18.3% (70 of 382) percutaneous umbilical cord sampling (PUBS) was performed; in 5.2% (20 of 382) cardiocentesis was performed to obtain fetal blood. RESULTS: Multivariate analysis showed an increase in odds of fetal loss for umbilical cord and cardiocentesis groups compared with the IHV FBS group. It was statistically significant (p < 0.01) only in the cardiocentesis group for fetal loss at < 2 weeks of performing the procedure.

Female↗
PubMed1998

Polymorphisms of twelve short tandem repeat loci in a Taiwanese population and their application in parentage testing.

With the advancement of techniques in molecular biology, rapid, sensitive, and reliable methods of DNA typing for parentage testing have become available. In this study, we evaluated the usefulness of multiplex polymerase chain reaction (PCR) with 12 unlinked short tandem repeat (STR) loci for paternity testing in Taiwan. The genetic informativeness of this test was then compared with that of conventional human leukocyte antigen (HLA) analysis in 167 parentage studies. The 12 STR loci alone provided a cumulative power of exclusion of up to 0.9998. Paternity was excluded in 59 (35.3%) cases, including 40 of 112 paternity trios and 19 of 55 paternity duos. In the 40 trios in which paternity was excluded, a mean of 6 (range, 3-9) incompatible STR markers were in the 19 duos in which paternity was excluded, a mean of 4 (range, 1-8) incompatible STR markers were noted. In the 72 trios in which the alleged paternity could not be excluded, the mean probabilities of paternity (PP) were 90.6863% with HLA testing alone, 99.9847% with STR analysis alone, and 99.9972% with combined HLA and STR analysis. In the 36 duos in which the alleged paternity could not be excluded, the mean PPs were 81.4768% with HLA testing alone, 99.6124% with STR analysis alone, and 99.9145% with combined HLA and STR analysis. These results suggest that STR analysis is very powerful when used alone for paternity trio testing and when combined with conventional serologic HLA typing for duo parentage testing in the Taiwan population.

Chromosome Mapping↗
PubMed1997

Specific inhibition of delta antigen by in vitro system by antisense oligodeoxynucleotide: implications for translation mechanism and treatment.

Synthetic antisense oligodeoxynucleotides (ODNs) and a system containing transcription and translation coupled rabbit reticulocyte lysate were used to develop a new model modulating the synthesis of small delta antigen which, in turn, inhibits the replication of HDV (hepatitis D virus). The ODN was stable for at least 50 min in this system at 37 degrees C. Unmodified 15-mer antisense D3 and D4, complementary to translation initiation region and coding region, respectively, inhibit the synthesis of small delta antigen by 95% at a concentration of 5 microM, whereas antisenses complementary to 5' noncoding region, stop codon region and polyadenylation site were less effective. This system also showed a dose-dependent inhibitory effect of antisense D3 on the production of the target protein. However, the synthesis of E6 protein, an internal control, was not affected. These observations imply that this in vitro system is convenient for rapid screening of effective antisense compounds and offers a promising perspective for the investigation of translation mechanisms and for the inhibition of HDV replication by antisense strategy.

Animals↗
PubMed1996

Secretory production of bioactive recombinant human granulocyte-macrophage colony-stimulating factor by a baculovirus expression system.

The proliferation and differentiation of hematopoietic cells are stimulated by a group of glycoproteins called colony stimulating factors (CSFs). Previously, we found that the human hepatoma cell line HA22T/VGH secreted a high level of human granulocyte-macrophage colony-stimulating factor (hGM-CSF). The cDNA of hGM-CSF, including the signal peptide sequence, was amplified from the total RNA of HA22T/VGH by a reverse-transcription polymerase chain reaction and was cloned into the pUC18 vector. After confirming the nucleotide sequence, the cDNA was inserted into a pVL1393 baculovirus transfer vector. The recombinant baculovirus carrying hGM-CSF cDNA was generated by co-transfecting the hGM-CSF recombinant transfer vector and BaculoGold baculovirus DNA into the Sf9 insect cells. The expected hGM-CSF transcript was detected in the recombinant virus-infected Sf9 cells. The conditioned media of the infected cells were analyzed by a slot-blot immunoassay. The results indicate that the infected insect cells produced and secreted hGM-CSF. According to colony forming assay, a maximum titer of 2.1 x 10(6). U ml-1 of hGM-CSF in the medium was obtained on the third day after infection.

Amino Acid Sequence↗
PubMed1996

Molecular analysis of cellular loci disrupted by papillomavirus 16 integration in cervical cancer: frequent viral integration in topologically destabilized and transcriptionally active chromosomal regions.

To discern the structural features of cellular loci that are disrupted by type 16 human papillomavirus (HPV-16) integration in cervical cancer, a polymerase chain reaction (PCR)-based strategy was employed for direct amplification and sequence analysis of four such cellular loci in cancer biopsy samples. One of the HPV-16-disrupted loci was found to be the microtubule-associated protein (MAP-2) gene and the other three loci were uncharacterized and were designated PID-1 to -3 (for papillomavirus integration-disrupted). The junctional sequences of the viral integration sites in the four loci analyzed are bracketed by long tracts of homogeneous purine or pyrimidine or alternating purine-pyrimidine which are known to destabilize the B-form conformation of the DNA structure. Using a panel of human/hamster hybrid cell DNAs and PCR analysis, the four loci were assigned to chromosomes 2 (MAP-2), 9 (PID-1), 1 (PID-2) and 8 (PID-3), respectively. These chromosomes carry numerous other previously determined viral integration and chromosomal fragile sites and the myc oncogenes. The PID-1 locus was further found in Southern analysis to be rearranged and amplified in another cervical cancer biopsy and a cervical carcinoma cell line (CaSki). On Northern analysis, the PID-1 and -3 probes detected a 3.0- and a 3.6-kb transcript, respectively, in normal cervical cells and in cervical cancer cell lines. The findings suggest that HPV-16 genome integrates frequently into topologically destabilized and transcriptionally active chromosomal sites. It remains to be elucidated whether the MAP-2 and the PID loci contribute to the pathogenesis of cervical cancer.

Base Sequence↗
PubMed1996

Prevalence and type of precore hepatitis B virus mutants in hepatitis D virus superinfection and its clinical implications.

To study the prevalence and type of the hepatitis B e antigen (HBeAg)-negative mutant in hepatitis D virus (HDV) superinfection, the precore region of hepatitis B virus (HBV) was analyzed by cycle sequencing. Of the 58 samples sequenced, 24 were wild type and 34 carried mutants. The precore stop mutation (TAG) at the 28th codon was found in 32 cases, other mutations were found in 7, and double mutations were found in 5. The absence of HBeAg showed a substantial agreement with the presence of mutants (kappa value, 0.74). Of the acute hepatitis patients, HDV replication and clinical manifestations were not significantly different between those with mutant and wild type virus, except that those with mutant virus were older (mean age, 48 vs. 28 years; P < .002). The absence of HBeAg in these patients is mainly due to HDV superinfection in older HBV carriers who already had precore mutant.

Acute Disease↗
PubMed1996

A method for screening antisense oligodeoxyribonucleotides effective for mRNA translation-arrest.

A transcription and translation coupled reticulocyte lysate system was established for rapid screening of antisense oligodeoxyribonucleotides (ODNs) to determine which are most effective for mRNA translation-arrest. A plasmid containing the target cDNA under the control of the T7 (or SP6) promoter was added to the lysate system in the presence of the T7 (or SP6) RNA polymerase, RNase H, and the antisense ODN under test. Transcription and translation were accomplished in a one-tube reaction. Translation-arrest caused by antisense ODN was evaluated in terms of the amounts of de novo-synthesized, [35S]-methionine or [35S]cysteine labeled target protein measured by gel electrophoresis and autoradiography. The properties of this system and optimal reaction conditions for use in antisense ODN screening were determined. Our method is simpler and more rapid than other in vitro screening methods.

Base Sequence↗
PubMed1996

Direct molecular diagnosis of CYP21 mutations in congenital adrenal hyperplasia.

The majority of congenital adrenal hyperplasia (CAH) cases arise from mutations in the steroid 21-hydroxylase (CYP21) gene. Without reliance on HLA gene linkage analysis, we have developed primers for differential polymerase chain reaction (PCR) amplification of the CYP21 gene and the non-functional CYP21P gene. Using the amplification created restriction site (ACRS) approach for direct mutational detection, a secondary PCR was then performed using a panel of primers specific for each of the 11 known mutations associated with CAH. Subsequent restriction analysis allowed not only the detection but also the determination of the zygosity of the mutations analysed. Existing deletion of the CYP21 gene could also be detected. In the analysis of 20 independent chromosomes in 11 families of CAH patients in Taiwan, four CYP21 mutation types, besides deletion, were detected. Interestingly, in five different alleles, the CYP21P pseudogene contained some polymorphisms generally associated with the CYP21 gene. These results suggest gene conversion events that are occurring in both CYP21P and CYP21 genes. Our combined differential PCR-ACRS protocol is simple and direct and is applicable for prenatal diagnosis of CAH using chorionic villi or amniotic cells.

Adrenal Hyperplasia, Congenital↗
PubMed1996

Constitutive production of colony-stimulating factors by human hepatoma cell lines: possible correlation with cell differentiation.

A panel of two poorly differentiated (HA22T/VGH and SK-Hep-1) and six well-differentiated (HuH-6-cl 5, HuH-7, PLC/PRF/5, Hep G2, Hep 3B, and Tong) human hepatocellular carcinoma (HCC) cell lines were studied for the production of colony-stimulating factors (CSFs) using the granulocyte and macrophage colony formation (CFU-GM) assay, immunocytochemical staining, and Northern blotting. Medium conditioned by untreated HA22T/VGH cells contained a high level of CSFs that could stimulate the in vitro colony formation of human myeloid progenitor cells. The HA22T/VGH cell-derived CSF had an apparent molecular weight of 23 kD. Its activity could be effectively neutralized by antiserum against granulocyte-macrophage CSF (GM-CSF) but not by antibodies to other hematopoietic growth factors, including G-CSF, M-CSF, interleukin-3 (IL-3), and IL-6. Correspondingly, immunocytochemical studies using monoclonal anti-GM-CSF showed a strong positive reaction in the cytoplasm of the HA22T/VGH cells. Northern blot analysis revealed that untreated HA22T/VGH cells expressed a considerable amount of GM-CSF mRNA, confirming that GM-CSF production was constitutive. At optimal concentrations, lipopolysaccharide (LPS), IL-1beta, interferon-gamma (IFN-gamma), and tumor-promoting phorbol diester (TPA) could all stimulate HA22T/VGH cells to secrete GM-CSF. In addition to HA22T/VGH, SK-Hep-1 cells could also produce GM-CSF, although less effectively, whereas all the well-differentiated HCC cell lines tested were negative for CSF production. Morphologic, cytochemical, and immunocytochemical examinations demonstrated that both poorly differentiated CSF-producing HCC cell lines (HA22T/VGH and SK-Hep-1) were macrophage-like in morphology, possessed nonspecific esterase (NSE) activity, and expressed CD14, CD68, and HLA-DR on their surface, while all the well-differentiated HCC cell lines were epithelioid and lacked myeloid differentiation antigens. These results suggest that monocytoid features and CSF production may be differentiation markers of hepatocytes at the immature stages, amd that the HA22T/VGH and SK-Hep-1 cell lines may be valuable tools for the study of hepatic function and differentiation.

Blotting, Northern↗
PubMed1996

Single-step hybridization screening for recombinant DNA clones with correct insert orientation and intact junction using a junctional oligonucleotide probe.

Cloning of DNA fragments with blunt ends or identical protruding ends results in colonies with the insertion sequence existing in two possible orientations. The orientation and the junctional sequence of the positive clones need to be established by means of restriction analysis and/or sequencing. Here, we proposed a rapid one-step method for the screening of clones not only with the desired orientation, but also with an intact junctional sequence. In this method, a 16-18 meric oligonucleotide probe synthesized according to the expected vector-insert junctional sequence is used as a probe in colony hybridization screening. Using this strategy, only recombinant clones which fulfill the above criteria will show positive signals in hybridization.

Base Sequence↗