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W Bi

Publications and source records attributed to W Bi.

36 records · Page 2Linked to original sources
PubMed1998

Detection of known mutation by proof-reading PCR.

Proof-reading PCR (PR-PCR) is designed to detect known mutations within genomic DNA. It differs from standard PCR approaches in that one of the two primers has its 3' end aligned with a putative mutation site, and has its 3'-OH replaced by a blocking group. Distinguishing a mutant gene from wild-type depends upon preferential removal of the blocked 3' terminal nucleotide by the polymerase proof-reading activity when it is mismatched with the template. Preferential removal of the blocked nucleotide allows subsequent extension and selective amplification, and provides the basis for distinguishing mutant from normal genes. This method has been used here to detect a transition mutation within the P53 gene of HaCaT cells with verification by direct sequencing of the selectively amplified DNA.

Carcinoma, Squamous Cell↗
PubMed1998

Detection of minimal residual disease in patients with AML1/ETO-associated acute myeloid leukemia using a novel quantitative reverse transcription polymerase chain reaction assay.

The AML1/ETO fusion transcript can be detected by reverse transcription polymerase chain reaction (RT-PCR) in patients with t(8;21)-associated acute myeloid leukemia (AML) in long-term complete remission (CR). Quantitation of the amount of the fusion transcript during CR may therefore be more predictive of cure or relapse than a simple qualitative assessment. Real Time PCR, a fluorometric-based technique, allows simple and rapid quantitation of a target sequence during the extension phase of PCR amplification, in contrast to end-point quantitative methods. Six patients with t(8;21)(q22;q22) AML, who achieved CR were studied by Real Time RT-PCR at different time intervals following diagnosis and high-dose cytarabine and anthracycline-based induction therapy. Five patients had a diagnostic bone marrow (BM) sample available for molecular analysis. Each patient showed > or = 10(3) copies of the AML1/ETO fusion transcript at diagnosis, and each showed a 2- to 4-log decrease in copy number following successful induction chemotherapy. This is comparable to the log-fold reduction in leukemic blasts that is thought to occur in patients successfully cytoreduced into CR by induction chemotherapy. The sixth patient showed a relatively high copy number immediately following successful remission induction chemotherapy, which continued to increase during early CR and was later followed by relapse. Real Time RT-PCR appears to offer advantages over previously used quantitative RT-PCR methods by providing absolute quantitation of the target sequence, expanding the dynamic range of quantitation to over six orders of magnitude, eliminating the post-PCR processing, and reducing labor and carryover contamination. These features make this an attractive method to prospectively evaluate the prognostic value of AML1/ETO fusion transcript quantitation in a larger patient population with t(8;21)(q22;q22) AML in CR.

Acute Disease↗
PubMed1998

Localization of mouse hepatitis virus open reading frame 1A derived proteins.

We have investigated the intracellular localization of proteolytic cleavage products encoded in the 5' portion of mouse hepatitis virus (MHV) gene 1. Immunofluorescent labeling of cells with an antiserum which recognizes p28, the ORF1a N-terminal cleavage product, resulted in widespread somewhat granular cytoplasmic staining, indicating that this protein is widely distributed in the cytoplasm of MHV-infected, but not control uninfected cells. Immunofluorescent staining of infected cells with antisera which recognize the downstream polypeptides, p65, p240 and p290 labeled discrete vesicular perinuclear structures. Double immunofluorescent labeling of BHK cells expressing the MHV receptor (BHK(MHVR1)) and infected with MHV-A59 with a Golgi-specific anti-mannosidase II monoclonal antibody and with antiserum recognizing each of these anti-MHV ORF1a polypeptides, showed that the p240 and p290 polypeptides were localized in discrete vesicular structures that overlapped the Golgi complex. Labeling with antibodies specific for p65 colocalized with the Golgi region, and showed staining of the perinuclear cytoplasm as well. Plasmids containing sequences contained in the first 6.75 kb of ORF1a have been expressed using the coupled vaccinia virus-T7 polymerase system. Immunofluorescent labeling of transfectants with the anti-ORF1a antisera showed patterns of antigen distribution similar to those observed in cells infected with MHV-A59. A deletion analysis with constructs containing only portions of the ORF1a sequence indicated that 303 amino acids containing the first papain-like protease domain (PLP-1) was sufficient to associate this protein with the Golgi.

Animals↗
PubMed1997

DNA binding specificity of the CCAAT-binding factor CBF/NF-Y.

CBF is a heterotrimeric protein that binds to DNA containing CCAAT motifs. Here we have analyzed interactions of recombinant CBF with DNA using hydroxyl radical footprinting and methylation interference assays. In the CBF-DNA complex, three separate DNA regions are protected from hydroxyl radical cleavage, one located over and immediately adjacent to the CCAAT motif itself and the other two located on both sides of the CCAAT motif. The methylation interference assay showed, however, that only in the CCAAT motif region methylation of bases was able to interfere with the formation of a CBF-DNA complex, suggesting that CBF makes sequence-specific contacts only in the CCAAT motif region. To further determine the specific DNA sequences necessary for CBF binding, we employed a polymerase chain reaction-mediated random binding site selection method. This analysis showed that CBF binding to DNA requires the CCAAT sequence and other specific sequences immediately flanking both ends of the CCAAT motif. We also showed that the nature of the flanking nucleotide sequences affects the affinity of CBF for DNA. Interestingly, most of the CCAAT motifs present in various higher eukaryotic promoters correspond to the CBF binding sites that were selected, consistent with the hypothesis that these motifs are binding sites for CBF and, hence, that CBF could regulate transcription of numerous eukaryotic genes.

Base Sequence↗
PubMed1997

CCR: a rapid and simple approach for mutation detection.

We describe a simple approach for detecting known mutations in genomic DNA. The strategy entails a DNA amplification reaction that combines the use of thermostable DNA polymerase and ligase, and that has been designated the Combined Chain Reaction (CCR). CCR consists of four phases: denaturation, annealing, elongation and ligation. Unlike most PCR-based mutation detection systems it relies on mismatch between primer and template at the primer 5'ends. It is rapid and simple, and requires neither the use of radioactivity, nor polyacrylamide gel electrophoresis, nor autoradiography for mutation detection at the single base-pair level.

DNA↗
PubMed1997

[CT analysis of hepatic metastases].

OBJECTIVE: To describe the manifestations of the hepatic metastases. To investigate the value of CT examination in diagnosis of hepatic metastases. METHOD: CT radiographs of 90 patients with hepatic metastases were studied retrospectively. The tumor number, size, density, shape, spreading in the hepatic parenchyma were analyzed. The size of liver and spleen was measured. RESULTS: Three or more metastatic nodules spreading in the hepatic parenchyma were found in 65 (72%) cases. Two nodules were shown in 6 (7%) cases. 19(21%) cases had only single nodule. 29 of the metastatic deposits located in the hepatic parenchyma near the liver edge. The size of metastatic nodules varied from 0.5 cm to 14 cm. In 87 cases, the density of metastatic nodules was lower than the surround liver parenchyma. In 3 cases of diffuse fatty liver, the metastatic deposits appeared as higher CT density nodules. Central necrosis of the tumor foci was seen in 44 cases. Striking hepatomegaly was noted in 31 cases with widespread nodules. In 26 cases which had one and two nodules, the shape of the liver was normal. In 45 cases, the primary tumour was found by CT at the same time. CONCLUSION: The CT findings of hepatic metastases differ. Multiple nodules disseminating in liver parenchyma is the general sign. Tiny nodules without other clinical signs can be detected by CT.

Adult↗
PubMed1996

Myocyte enhancer binding factor-2 expression and activity in vascular smooth muscle cells. Association with the activated phenotype.

Proliferation and phenotypic modulation of smooth muscle cells (SMCs) are major components of the vessel's response to injury in experimental models of restenosis. Some of the growth factors involved in restenosis have been identified, but to date little is known about the transcription factors that ultimately regulate this process. We examined the expression of the four members of the myocyte enhancer binding factor-2 (MEF2) family of transcription factors in cultured rat aortic SMCs (RASMCs) and a rat model of restenosis because of their known importance in regulating the differentiated phenotype of skeletal and cardiac muscle. In skeletal and cardiac muscle, the MEF2s are believed to be important for activating the expression of contractile protein and other muscle-specific genes. Therefore, we anticipated that the MEF2s would be expressed at high levels in medial SMCs that are producing contractile proteins and that they would be downregulated along with the contractile protein genes in neointimal SMCs. On the contrary, we observe that MEF2A, MEF2B, and MEF2D mRNAs are upregulated in the neointima, with the highest levels in the layer of cells nearest to the lumen, whereas MEF2C mRNA levels do not appreciably increase. Moreover, few cells in the media are making MEF2 proteins detectable by immunohistochemistry, whereas large numbers of neointimal cells are positive for all four MEF2s. These data suggest that the MEF2s are involved in the activated smooth muscle phenotype and not in the maintenance of contractile protein gene expression.

Animals↗
PubMed1996

The glutathione S-transferase M1 (GSTM1) null genotype and benzidine-associated bladder cancer, urine mutagenicity, and exfoliated urothelial cell DNA adducts.

Multiple studies in the general population have suggested that subjects with the glutathione S-transferase M1 (GSTM1)-null genotype, who lack functional GSTM1, are at higher risk for bladder cancer. To evaluate the impact of the GSTM1-null genotype on bladder cancer caused by occupational exposure to benzidine and to determine its influence on benzidine metabolism, we carried out three complementary investigations: a case-control study of bladder cancer among workers previously exposed to benzidine in China, a cross-sectional study of urothelial cell DNA adducts and urinary mutagenicity in workers currently exposed to benzidine in India, and a laboratory study of the ability of human GSTM1 to conjugate benzidine and its known metabolites in vitro. There was no overall increase in bladder cancer risk for the GSTM1-null genotype among 38 bladder cancer cases and 43 controls (odds ratio, 1.0; 95% confidence interval, 0.4-2.7), although there was some indication that highly exposed workers with the GSTM1-null genotype were at greater risk of bladder cancer compared to similarly exposed workers without this allele. However, the GSTM1 genotype had no impact on urothelial cell DNA adduct and urinary mutagenicity levels in workers currently exposed to benzidine. Furthermore, human GSTM1 did not conjugate benzidine or its metabolites. These results led us to conclude that the GSTM1-null genotype does not have an impact on bladder cancer caused by benzidine, providing a contrast to its association with elevated bladder cancer risk in the general population.

Benzidines↗
PubMed1995

Intracellular localization of polypeptides encoded in mouse hepatitis virus open reading frame 1A.

We have investigated the intracellular localization of several of the proteolytic cleavage products derived from the 5' portion of mouse hepatitis virus (MHV) gene 1. Antisera UP1 recognizes the N-terminal ORF1a cleavage product p28. Immunofluorescent staining of cells with this antisera resulted in a diffuse punctate pattern of cytoplasmic staining, indicating that this protein is widely distributed in the cytoplasm. Immunofluorescent staining of infected cells with antisera which recognize polypeptides p240 and p290 stained discrete vesicular perinuclear structures suggesting that these proteins localized to the Golgi. This was confirmed by double immunofluorescent staining of BHK cells expressing the MHV receptor (BHK-R) with a Golgi specific antibody in addition to our anti-MHV ORF1a antibodies. Antisera UP102 recognizes p28 and the immediately downstream p65 gene product. Double immunofluorescent staining of MHV infected BHK-R cells with UP102 labeled discrete vesicular structures overlapping the Golgi complex. In addition there was punctate staining more widely distributed in the cytoplasm. The simplest explanation for this pattern is that p65 is also localized to the Golgi region of the cell, whereas p28 is more widespread. Plasmids containing the first 4.7 and 6.75 kb of ORF 1a have been expressed using the coupled vaccinia virus-T7 polymerase system. Images obtained by immunofluorescent staining of transfectants with our anti-ORF1a antisera are similar to those obtained during infection with A59. These studies indicate that the signals which direct p290 to the Golgi are likely contained between the C-terminus of p28 and ORF1a residue 1494.

Animals↗
PubMed1995

The truss structure of cancellous bone. Morphological basis of the function of load transmission of the synovial joint.

The structure of cancellous bone of synovial joints was studied under the scanning electron microscope in 4 young cadavers. It was discovered that in all specimens, the cancellous bone beneath the whole coverage of the articular cartilage had a honeycomb pattern in appearance formed by many arched trabeculae running in different directions, and that the major orientation of the arched trabeculae was toward the articular surface. The arms of one arched trabecula extended in different directions, forming the tops of other arched trabeculae; the direction of the collagen fibers conformed circumferentially with that of the arch, but the collagen fibers at the intermediate part of the common arm of the adjacent arched trabeculae crossed in a woven pattern, passing from one trabecula to another. It enables the whole end of the articular bone to have the capacity of integral deformation. That is the foundation of the character of compliance which is essential to the contact of the articular surfaces changing from incongrouity to total congrouity during normal load transmission. This special type of construction is just like the truss structure in the architectural engineering, and therefore the authors suggest to name it the "Truss Structure" of the cancellous bone. The relationship between integral deformation and the following two factors, compliance and incongrouity of the articular surface provides an explanation that the truss structure of the cancellous bone is the morphological basis of high load-bearing capacity and character of compliance of the synovial joint.

Adult↗
PubMed1994

Mouse hepatitis virus gene 5b protein is a new virion envelope protein.

Highly purified radiolabeled mouse hepatitis virus (MHV) A59 contained a previously overlooked protein which coelectrophoreses with the gene 5b product immunoprecipitated from infected cells. The gene 5b protein is post-translationally acylated. Rabbit antibody raised against a recombinant gene 5b protein expressed in Escherichia coli neutralized viral infectivity in the presence of complement, although not in the absence of complement. Immunofluorescent staining of MHV-infected cells with two anti-peptide antibodies revealed that the gene 5b product is membrane-associated and is transported to the cell surface, findings consistent with the prediction of a membrane-spanning segment in the gene 5b polypeptide. These results suggest strongly that the gene 5b polypeptide represents a new MHV virion envelope protein which is homologous to the TGEV ORF 4 and IBV 3c proteins.

Antigens, Viral↗
PubMed1993

N-acetylation phenotype and genotype and risk of bladder cancer in benzidine-exposed workers.

Several studies in subjects occupationally exposed to arylamine carcinogens have shown increased risks for bladder cancer associated with the slow acetylator phenotype. To follow up these reports, a case-control study of N-acetylation and bladder cancer risk was carried out among subjects occupationally exposed to benzidine, in benzidine dye production and use facilities in China. Thirty-eight bladder cancer cases and 43 controls from these factories were included for study of acetylation phenotype, by dapsone administration, and for polymorphisms in the NAT2 gene, by a polymerase chain reaction (PCR)-based test. In contrast to previous studies, no increase in bladder cancer risk was found for the slow N-acetylation phenotype (OR = 0.3; 95% CI = 0.1-1.3) or for slow N-acetylation-associated double mutations in NAT2 (OR = 0.5; 95% CI = 0.1-1.8). Examination of specific mutations and adjustment for age, weight, city and tobacco use did not alter the results. When examined by level of benzidine exposure in the cases, the bladder cancer risks associated with low (OR = 0.3, 95% CI = 0.0-2.2), medium (OR = 0.7, 95% CI = 0.1-4.5) and high (OR = 0.6, 95% CI = 0.1-3.5) exposure showed no interaction between genotype and benzidine exposure, within the range of exposures experienced by subjects in this study. This study, which is the first to incorporate phenotypic and genotypic analyses, provides evidence that the NAT2-related slow N-acetylation polymorphism is not associated with an increased risk of bladder cancer in workers exposed to benzidine, and may have a protective effect.

Acetylation↗
PubMed1993

Correlation between N-acetyltransferase activity and NAT2 genotype in Chinese males.

Eighty-four healthy Chinese male control subjects derived from an occupation-based case-control study of bladder cancer were evaluated for hepatic N-acetyltransferase activity by dapsone and for NAT2 genotype using allele-specific amplification of peripheral leukocyte DNA by the polymerase chain reaction. Fifty-nine percent of the overall variation in acetylation activity was explained by genotype (p < 0.0001). The remaining variation in acetylation was not associated with dapsone N-hydroxylation activity, age, current smoking status, or weight in the study population, or within any genotype subgroup. Although acetylation activity in the homozygous mutant group did not overlap with the other genotype categories, there was moderate overlap in acetylation between the heterozygous mutant and wildtype groups, and substantial variation in acetylation within them. Considering all subjects with the identical NAT2 genotype as phenotypically similar and all subjects with differing NAT2 genotypes as phenotypically distinct may result in misclassification of metabolic risk factors in epidemiological investigations. As such, it would seem prudent, where possible, to collect both acetylation phenotype and NAT2 genotype data, since the advantages and limitations of these two sources of information complement, and serve to assess the accuracy of each other.

Aged↗
PubMed1993

Field molecular epidemiology. Feasibility of monitoring for the malignant bladder cell phenotype in a benzidine-exposed occupational cohort.

Modern molecular and cellular biology have provided powerful new approaches to study cancer in the research laboratory, but these techniques have not been used extensively in field studies or in screening of high-risk occupational cohorts. The primary objective of this study was to demonstrate the use of cellular and molecular methods in combination with medical and epidemiologic methods to identify cancer cases, risk factors, and markers in a previously identified and defined cohort of Chinese workers exposed to benzidine. The screening of exposed workers included occupational, medical, and smoking histories to identify exogenous risk factors, a limited physical examination, Papanicolaou (PAP) urinary cytology, measurement of urine pH, quantitative fluorescence image analysis (QFIA) cytology to detect DNA hyperploidy, and quantitative fluorescence to detect expression of a low-grade bladder tumor-associated antigen (p300) by exfoliated urothelial cells and elevated expression of the neu oncogene product (p185). Detailed analysis of the accuracy of epidemiologic data and the adequacy of samples and accuracy of molecular techniques was carried out. Three groups were included in this study: group 1 included 23 bladder cancer cases who had previously been exposed to benzidine and served as two surrogates for late-emerging disease; group 2 consisted of 20 subjects with previous exposure history but not previously diagnosed with bladder cancer; and group 0 was the nonexposure and nonbladder cancer controls. The results showed that accurate questionnaire data and urine samples can be obtained at remote sites. Among the 20 group 2 subjects, two displayed abnormal findings by both QFIA cytology and p300 expression and were later confirmed to have bladder cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzidines↗
PubMed1992

Mortality and incidence of bladder cancer in benzidine-exposed workers in China.

We examined bladder cancer mortality and incidence to 1981 in 1,972 workers employed in benzidine-exposed jobs in Tianjin, Shanghai, and Jilin, China, between 1972 and 1977, and in 1,974 unexposed workers employed during the same time period. In comparison to general population rates, in the benzidine-exposed group the ratio of observed to expected deaths (SMR) was 17.5 (95% C.I.: 7.5-34.5) and the ratio of observed to expected incident cases (SIR) was 25.0 (95% C.I.: 16.9-35.7). No excess was noted in the unexposed group. The 25-fold increase in bladder cancer incidence in the exposed group was related to level of exposure, with the SIR rising from 4.8 for low exposure to 36.2 for medium exposure, and 158.4 for high exposure. Risks were elevated both for producers of benzidine (SIR = 45.7; 95% C.I.: 20.9-86.8) and for users (SIR = 20.9; 95% C.I.: 12.9-32.0) of benzidine dyes. Benzidine-exposed workers who smoked tobacco had a 31-fold risk (95% C.I.: 20.4-46.4), while non-smoking workers had an 11-fold risk (95% C.I.: 3.6-25.8), suggestive of a multiplicative relationship between these two carcinogens.

Air Pollutants, Occupational↗
PubMedDate not supplied

An HSVtk-mediated local and distant antitumor bystander effect in tumors of head and neck origin in athymic mice.

The "bystander effect," produced by ganciclovir-mediated killing of cells transduced with a herpes simplex virus thymidine kinase (HSVtk) gene, defines the cooperative killing of non-HSVtk-transduced cells. In vitro, a major contributor to this phenomenon is metabolic cooperation involving transfer of cytotoxic small molecules between cells via gap junctions. In this study, the bystander effect was assessed in vivo using cells of oral squamous cell carcinoma origin. Mixtures of HSVtk+ and HSVtk- tumor cells were implanted subcutaneously in the left flank of nude mice, and naive HSVtk- cells were implanted subcutaneously in the right flank. When tumors attained a size of 0.5 to 1 cm, the animals were treated with ganciclovir on a daily basis. The tumors comprised of mixed cells in the left flank resolved, consistent with a predicted bystander effect. The naive tumors in the right flank either resolved or became cytostatic showing little further growth compared to controls. Similar results were obtained when naive tumors were grown in both flanks and the tumor in the left flank received intratumoral injection of HSVtk retroviral producer cells or PA317 (HSVtk+) packaging cells, but not parental NIH 3T3 cells. Concomitant treatment with dexamethasone impaired the antitumor effect on the contralateral side. When these experiments were performed in SCID-Beige mice, there was a reduced antitumor effect on the ipsilateral flank and no antitumor response in the contralateral flank. Together with histology of regressing tumors, which showed an infiltration of lymphoid cells, these results are suggestive of an immune-related antitumor response that could account for the distant bystander effect.

3T3 Cells↗