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Biomedical subjects

M D Lai

Publications and source records attributed to M D Lai.

At least 37 records · Page 2Linked to original sources
PubMed1995

Detection of CYP2C9 polymorphism based on the polymerase chain reaction in Chinese.

Cytochrome P450 (CYP) 2C9 catalyses the metabolism of a wide range of drugs. Previous studies have shown the differences in the amino acid composition among CYP2C9 variants at Cys144/Arg, Tyr358/Cys, Leu359/Ile, and Gly417/Asp. PCR-endonuclease digestion methods have been developed to detect these four possible polymorphisms. The T416-->C mutation in exon 3 of CYP2C9 (Cys144-->Arg) creates an Ava II site. In the 135 subjects we tested, all leukocyte DNA samples showed a complete Ava II digestion indicating homozygous C416 (Arg144). A Tyr358-->Cys mutation will create a Nsi I site at codon 1057-1063 in exon 7. In 40 subjects tested, all samples showed negative results. DNA sequencing on a few samples showed Tyr358Ile359. A mismatched PCR primer pair was then designed to detect codon C1061-->A (Leu359-->Ile) mutation. In 115 subjects tested, 111 samples showed a complete Nsi I digestion (Ile359) and four samples showed heterozygous results. Another mismatched PCR primer pair was used to confirm the C1061 codon in heterozygous subjects. The four heterozygous subjects showed partial digestion with endonuclease Kpn I, which confirmed the heterozygous Ile/Leu at amino acid 359. The G1236-->A mutation in exon 8 of CYP2C9 (Gly417-->Asp) creates a Hph I site. In all 46 subjects, homozygous G1236 (Gly417) was found. Most Chinese subjects actually have Arg144 Tyr358 Ile359 Gly417 in CYP2C9 as previously reported human-2. Furthermore, we found an A-->T (+12 position in intron 2) mutation in our CYP2C9 sequencing process. The mutation creates a NIa III site.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1995

Population genetic study of selected tetranucleotide repeat DNA polymorphisms on chromosomes Y and 12.

Microsatellite polymorphisms of 75 unrelated Taiwanese males were analyzed for (GATA) repeats in DYS19 on chromosome Y and tetranucleotide simple repeats in D12S67 on chromosome 12. A simple nonisotopic technique based on polymerase chain reaction electrophoresis on native polyacrylamide gels followed by silver staining was used. In the 75 unrelated males, four alleles on DYS19 and five alleles on D12S67 were detected. Different allele frequencies, heterozygosity rates and information on the nature of the repeat loci were determined in these unrelated individuals. The resulting polymorphisms are of great assistance in ethnologic studies, forensic investigation and deficiency cases in paternity testing.

Base Sequence↗
PubMed1995

Analysis of K-ras gene mutations in periampullary cancers, gallbladder cancers and cholangiocarcinomas from paraffin-embedded tissue sections.

Point mutations of the K-ras gene were analyzed in 25 periampullary cancers (21 ampulla vater cancers, two common bile duct cancers and two duodenal cancers), two gallbladder cancers and six cholangiocarcinomas. DNA extracted from the paraffin-embedded tissues was amplified with the polymerase chain reaction and subsequently analyzed by direct cycle sequencing at codons 12, 13 and 18 of the K-ras gene. Codon 61 was first screened with single strand conformation polymorphism and then sequenced by direct cycle sequencing. No point mutation was found in any of the 25 periampullary cancers or the two gallbladder cancers. These results are similar to previous reports. Mutation of the K-ras gene seems not to play an important role in tumorigenesis of periampullary cancer. In two of six (33%) cholangiocarcinoma patients, point mutations were found. Both mutations were transitions, GGT to GAT at codon 12. The incidence of mutation was greater than that in Thailand (about 8%) but less than that in Japan (about 60%). Mutation of the K-ras gene may play varied roles in the tumorigenesis of cholangiocarcinoma, depending on geographic area.

Adult↗
PubMed1994

[Immunohistochemical study of neuroendocrine cells and prostate-specific antigen in prostate carcinoma].

In 21 cases of prostate adenocarcinoma and 4 cases of prostate hyperplasia, neuroendocrine (NE) cells were studied immunohistochemically with anti-chromogranin antibody. NE cells were detected in 8 cases (38.8%), in which 4 were found positive for serotonin and 2 for glucagon. 25 cases of prostatic specimens and 7 cases of nonprostatic adenocarcinomas were labelled with prostate-specific antigen (PSA). It was shown that expression of PSA was closely correlated with differentiation of prostatic adenocarcinomas. Reaction of PSA was stronger in well-moderately differentiated adenocarcinoma than in poorly differentiated one. For staining pattern, positive reaction of PSA was located in the apical border in well-moderately differentiated adenocarcinoma, but in the cytoplasm and cell membrane in poorly differentiated one.

Adenocarcinoma↗
PubMed1993

Partial reversion of transformed phenotype of B104 cancer cells by antisense nucleic acids.

We used antisense RNA to inhibit the expression of oncogene neu and investigated the effects of diminished neu expression on the phenotypes of B104 cells containing activated oncogene neu. Antisense MT-neu and pSV-neo plasmids were cotransfected into neuroblastoma B104 cells. Southern analysis showed the integration of anti-neu DNA into B104 cells. The expression of neu was inhibited up to 90% as quantitated by immunoprecipitation. The growth rate and the potential to differentiate in these transfectants were not affected as compared to the parental cell lines. The ability to grow in soft agar was inhibited more than 90% in these transfectants. Our results indicated that antisense-RNA against a specific oncogene can decrease the tumorigenicity of tumor cells but may not be able to revert it to normal cells completely.

Animals↗
PubMed1993

Release of the p53-induced repression on thymidine kinase promoter by single p53-binding sequence.

Herpes simplex thymidine kinase (TK) promoter was shown to be repressed by the wild-type p53. Using a model system that the p53-binding site was linked to the thymidine kinase promoter, we demonstrated that single p53-specific binding site was sufficient to abolish the repression. On the contrary, the mutant p53 had the opposite effects on the HSV-TK promoter in BHK cells. The results suggest that the p53-binding site may act as an enhancer to regulate the gene expression in a novel way in vivo.

Animals↗
PubMed1993

Molecular basis of genetic variation in debrisoquin hydroxylation in Chinese subjects: polymorphism in RFLP and DNA sequence of CYP2D6.

Debrisoquin hydroxylation phenotype was determined in 124 Chinese persons living in Taiwan, and two poor metabolizers were identified with a urinary metabolic ratio (MR) greater than 12.6. The other subjects, extensive metabolizers, showed a normal frequency distribution of log(MR). Most subjects (50%) showed a 44/29 kb pattern in restriction fragment length polymorphism (RFLP) analysis with use of Xba I, and 30% and 15% of the subjects exhibited a homozygous 29/29 kb and 44/44 kb pattern, respectively. Among extensive metabolizers, subjects with the 44/44 kb pattern had a significant higher log(MR) than those with the 44/29 pattern, and the log(MR) of the subjects with the 44/29 kb pattern was significantly higher than that of the subjects with 29/29 kb pattern. All nine exons and intron 3 of C gamma P2D6 were amplified with polymerase chain reaction (PCR) and sequenced for four extensive metabolizers. Two major polymorphisms were found: one at position 188 of exon 1 and the other at position 4268 in exon 9. With PCR and endonuclease digestion, polymorphisms at exon 1, intron 3, and exon 9 were investigated. Only two of 254 alleles showed a heterozygous guanine at 1934 base pairs (G1934) to adenine (A) mutation, commonly found in white poor metabolizers. Approximately 70% of alleles showed thymine at 188 base pairs (T188), and 76% showed cytosine at 4268 base pairs (C4268) instead of C188 and G4268, as is found in most white subjects. Subjects with T188 or C4268 showed a significant higher log(MR) than subjects with homozygous C188 and G4268. The C/T188, G/A1934, G/C4268, and RFLP polymorphisms may explain the interracial variations between Chinese and white subjects, as well as the genetic variations among Chinese subjects.

Adult↗
PubMed1992

Analysis of proliferative activity in colorectal mucosa by immunohistochemical detection of proliferating cell nuclear antigen (PCNA). Methodological aspects and application to routine diagnostic material.

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) has been suggested as a new approach for determining proliferative activity in paraffin-embedded tissue. In a prospective study PCNA immunostaining was performed in 284 colorectal biopsies using monoclonal antibodies 19F4 (Ogata et al. 1987) and PC10 (Waseem and Lane 1990) and compared with the Ki67 method. From each site three biopsies were taken and a variety of fixation regimens for frozen and paraffin-embedded samples tested. For frozen biopsies methanol fixation at -20 degrees C proved best. In paraffin sections PCNA could be detected after methacarn fixation as well as after controled fixation at 4 degrees C in 4% paraformaldehyde for 1 h and in most biopsies routinely fixed with 10% formalin. However, the latter fixation regimens revealed additional PCNA-positive cells in the normal superficial colonic mucosal epithelium. Although the percentage of cells positive for PCNA was generally lower than for Ki67, the rates correlated in a highly significant fashion, both in frozen methanol-fixed biopsies, and in paraformaldehyde-fixed paraffin-embedded samples. PCNA immunohistochemistry revealed a similar proliferative activity in different parts of the large bowel. A higher proliferative activity was found in inflamed mucosa, adenomas, carcinomas and even in normal mucosa from patients with colorectal neoplasms. In routinely fixed biopsies, the monoclonal antibody PC10 was superior to 19F4 because of considerably less background staining. However, in the routine material only a rough estimate of the proliferative activity was possible by PCNA immunohistochemistry using these antibodies, because unpredictable numbers of non-S-phase cells were also stained.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Histopathology and immunohistopathology of lymph nodes of the first autopsy with HIV positivity in China.

Nineteen lymph nodes of a HIV-positive boy were studied histologically and immunohistologically. According to Stutte's classification of HIV-related lymphadenopathy, 84% of the lymph nodes were at the third or fourth stages in relation to clinical status ARC/AIDS. Lymph follicle atrophy, angiogenesis, histiocytic proliferation and destruction of the normal reticulum frame were observed. Immunohistochemical studies showed most of the remaining lymphocytes to be T cells and changes in the distribution of S-100 positive cells. Ki 1 positive cells existed mainly at the second stage. The significance of these changes is discussed.

Antigens, CD↗
PubMed1991

The effects of glucocorticoid hormone on the expression of c-jun.

The effects of glucocorticoid hormone on the expression of c-jun in the fibroblasts were studied. The expression of c-jun was repressed by dexamethasone in the NIH3T3 cells, but not in the transformed B104-1 or EJ-Ras cells. The repression was not relieved by the addition of cycloheximide.

Animals↗
PubMed1991

C-myc expression in early human placenta--a critical evaluation of its localization.

A tight association between the expression of the protooncogene c-myc and the proliferation of trophoblast in first trimester human placentae has been reported, supporting the view that c-myc is under close control of the cell cycle. However, this has not been verified in several other cells systems. Therefore we reexamined the exact localization of myc expression at the transcriptional and translational level in 20 first trimester and three term placentae. Myc mRNA and protein was sparse or absent at term but abundant in early gestation placentae. The proliferative cell columns and the villous cytotrophoblast contained the greatest amounts, revealing myc protein in around 60-70% of villous cytotrophoblast cells. Unexpectedly, a considerable fraction of the syncytiotrophoblast nuclei of early placentae (20%) also showed myc expression, and this was particularly evident on the protein level. The myc content estimated by immunohistochemistry decreased with increasing placental maturation. In addition, prominent myc expression was seen in decidual cells, suggesting a paracrine growth regulation of the gestational endometrium. Our findings do not support the notion that myc expression is closely cell cycle-dependent. On the contrary, it appears that in the human placenta, myc expression characterizes the phase of rapid organ development in the first trimester and is not restricted to the proliferative cytotrophoblast.

Alkaline Phosphatase↗
PubMed1988

Influence of DNA sequence on the nature of mispairing during DNA synthesis.

A series of synthetic oligonucleotide primers, annealed at various positions along the lacZ-alpha region of bacteriophage M13mp9 template, were elongated by purified DNA polymerases in the presence of only 3 of the 4 deoxynucleoside triphosphates to achieve misincorporation at a total of 49 different positions along the template. The newly synthesized strands (containing misincorporated bases) were isolated and sequenced to determine the identity of misincorporated deoxynucleoside monophosphates. The results indicate that the kind of mispairing that occurs during DNA synthesis is greatly influenced by the nucleotide sequence of the template. Transition-type base substitutions predominated overall, but at many template positions, transversion-type base substitutions occurred, most commonly via A.A mispairing. The results of parallel determinations made with Escherichia coli DNA polymerase I ("large fragment" form) and DNA polymerase of Maloney murine leukemia virus indicated that, overall, the identity of polymerase had only a small effect on the kind of misincorporation that occurred at different positions along the template. However, at certain template positions, the nature of mispairing during DNA synthesis was reproducibly affected by differing polymerase active-site environment.

Base Composition↗
PubMed1988

Influence of divalent metal activator on the specificity of misincorporation during DNA synthesis catalyzed by DNA polymerase I of Escherichia coli.

To test whether the identity of divalent metal activator affects the specificity of misincorporation during polymerization catalyzed by E. coli DNA polymerase I, we carried out the following procedure. A series of oligonucleotide primers, annealed at different positions along the lacZ region of bacteriophage M13mp9 DNA, were elongated in the presence of 3 of the 4 deoxynucleoside 5'-triphosphates (dNTPs) until one or a few misincorporations occurred in each elongated primer. The elongated primers (containing deoxynucleotide residues that had been misincorporated in the presence of either Mg2+ or Mn2+) were then isolated and sequenced by the 'dideoxy' chain termination method to determine the identity of deoxynucleoside monophosphates (dNMPs) that had been misincorporated at different template positions during the original 'minus' reactions, activated by Mg2+ or Mn2+. The results obtained by this approach revealed that both the type of misincorporation and the effect of substituting Mn2+ for Mg2+ depended on the nucleotide sequence of the template. At 40% of the template positions at which misincorporation was compared with both metal ions (8 out of 20), the identity of mispairs differed significantly for synthesis activated by Mn2+ versus Mg2+. Of these 8 sites, 4 exhibited increased transversions in the presence of Mn2+, while 4 exhibited decreased transversions with Mn2+.

Bacterial Proteins↗
PubMed1982

Role of aromatic residues in the structure-function relationship of alpha-bungarotoxin.

The conformation of alpha-bungarotoxin and its cyanogen bromide cleaved and nitrated derivatives was studied by circular dichroism (CD). Native toxin contains no helices but some beta forms and possibly beta turns. Its ordered conformation is little affected when the peptide bond between Met-27 and Trp-28 is cleaved; however, the CD due to Trp-28 is abolished. The CNBr-cleaved derivative retains its immunoaffinity toward anti-toxin sera but loses its neurotoxicity toward the acetylcholine receptor. On the basis of both CD and fluorescence spectra, Trp-28 is probably stabilized by a short-range interaction with the carboxylate group of Asp-30. The ordered conformation of the toxin is also unaltered when one of the two tyrosine residues, identified as Tyr-54, is nitrated with tetranitromethane. This Tyr(NO2)-54 derivative possesses both immunoaffinity and neurotoxicity. However, the toxin is denatured and loses its activities when the other tyrosine residue, Tyr-24, is also nitrated in 6 M guanidine hydrochloride, even after the denaturant is removed. Spectrophotometric titration of the toxin indicates that Tyr-54 has a normal apparent dissociation constant (pKa = 9.7) and Tyr-24 ionizes at pH above 11.2. Both tyrosine residues are in a polar environment, but Tyr-24 is not readily accessible to reagents and is stabilized by long-range interactions, probably involving Glu-41.

Amino Acid Sequence↗
PubMedDate not supplied

Microarray profiling of gene expression patterns in bladder tumor cells treated with genistein.

Microarray technology was used to gain an insight into the molecular events of tumor cell growth inhibition mediated by the soy isoflavone genistein. For this, a susceptible bladder tumor line TCCSUP was treated with the inhibitory dose (50 microM) of genistein for various periods of time, followed by mRNA isolations, cDNA probe preparations, and hybridization individually to cDNA chips containing 884 sequence-verified known human genes. After analyzing the hybridization signals with a simple quantitative method developed by this study, we detected that egr-1, whose expression has been associated with proliferation and differentiation, was transiently induced and this expression pattern was later confirmed by RT-PCR. Thus, microarray technology is a reliable and powerful tool for profiling gene expression patterns in many biological systems related to cancer. We further detected many groups of genes with distinct expression profiles and most of them encode for proteins that regulate the signal transduction or the cell cycle pathways. These genes warrant further investigation as regards their roles in the susceptibility of the tumor cell line to the antitumor drug.

Antineoplastic Agents↗