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Biomedical subjects

W L Kuo

Publications and source records attributed to W L Kuo.

66 records · Page 4Linked to original sources
PubMed1991

Clinical applications of fluorescence in situ hybridization.

We review here the application of fluorescence in situ hybridization with chromosome-specific probes to chromosome classification and to detection of changes in chromosome number or structure associated with genetic disease. Information is presented on probe types that are available for disease detection. We discuss the application of these probes to detection of numerical aberrations important for prenatal diagnosis and to detection and characterization of numerical and structural aberrations in metaphase spreads and in interphase nuclei to facilitate tumor diagnosis.

Chromosome Aberrations↗
PubMed1991

Molecular cytometry applied to detection and characterization of disease-linked chromosome aberrations.

Flow karyotyping and FISH with chromosome-specific or disease locus-specific probes are powerful adjuncts to conventional cytogenetic analysis. Flow karyotyping is well suited to quantitative analysis of the DNA content changes that occur during structural rearrangement. FISH with probes for repeated sequences allows ready detection of aneuploidy in interphase cells. FISH with whole-chromosome composite probes to metaphase spreads facilitates detection of subtle structural changes and allows detection of structural aberrations that occur at frequencies as low as 10(-3). FISH with locus-specific probes facilitates diagnosis of specific genetic diseases, may allow phenotype-genotype correlation on a cell by cell basis and will permit sensitive detection of residual disease.

Chromosome Aberrations↗
PubMed1991

Localization of the gene encoding insulin-degrading enzyme to human chromosome 10, bands q23----q25.

Insulin-degrading enzyme (IDE) is a cytosolic proteinase involved in the cellular processing of insulin. Using somatic cell hybrid analysis and in situ chromosomal hybridization, we have localized the gene encoding IDE to human chromosome 10, bands q23----q25. The murine Ide gene was previously mapped to Chromosome 19; together, these results suggest that the IDE gene is a member of a conserved syntenic group on human chromosome 10, bands q23----q25 and mouse Chromosome 19.

Animals↗
PubMed1991

Regulation of insulin degradation: expression of an evolutionarily conserved insulin-degrading enzyme increases degradation via an intracellular pathway.

The insulin-degrading enzyme (IDE) is an evolutionarily conserved enzyme that has been implicated in cellular insulin degradation, but its site of action and importance in regulating insulin degradation have not been clearly established. We addressed this question by examining the effects of overexpressing IDE on insulin degradation in COS cells, using both human IDE (hIDE) and its Drosophila homolog (dIDE). The dIDE, which was recently cloned in our laboratory, has 46% amino acid identity with hIDE, degrades insulin with comparable efficiency, and is readily expressed in mammalian cells. Transient expression of dIDE or hIDE in COS monkey kidney cells led to a 5- to 7-fold increase in the rate of degradation of extracellular insulin, indicating that IDE can regulate cellular insulin degradation. Insulin-degrading activity in the medium was very low and could not account for the difference between transfected and control cells. To further localize the site of IDE action, the fate of insulin after receptor binding was examined. The dIDE-transfected cells displayed increased degradation of prebound insulin compared to control cells. This increase in degradation was observed even when excess unlabeled insulin was added to block reuptake or extracellular degradation. These results indicate that IDE acts at least in part within the cell. The lysosomotropic agents chloroquine and NH4Cl did not affect the increase in insulin degradation produced by transfection with dIDE, indicating that the lysosomal and IDE-mediated pathways of insulin degradation are independent. The results demonstrate that IDE can regulate the degradation of insulin by intact cells via an intracellular pathway.

Ammonium Chloride↗
PubMed1991

Applications of fluorescence in situ hybridization in biological dosimetry and detection of disease-specific chromosome aberrations.

Dual color FISH with whole chromosome and pan-centromere probes facilitates rapid detection of stable structural aberrations such as translocations. This approach should allow analysis of translocations for assessment of genetic damage at long times after exposure or as a result of chronic exposure during a long period of time. Multi-color FISH with locus specific probes allows assessment of the frequency of cells carrying specific aberrations known to be associated with tumorigenesis, analysis of the series of genetic changes that occur during tumor evolution and correlation between genotype and phenotype. The power of FISH for analysis of random and tumor related events will increase steadily as informative probes are developed during the course of the International Human Genome Project.

Chromosome Aberrations↗
PubMed1991

Detection of aneuploidy involving chromosomes 13, 18, or 21, by fluorescence in situ hybridization (FISH) to interphase and metaphase amniocytes.

Fluorescence in situ hybridization (FISH) with chromosome-specific probes has been applied to detection of numerical aberrations involving chromosomes 13, 18, and 21 in metaphase and interphase amniocytes. High-complexity, composite probes for chromosomes 13, 18, and 21 were used as hybridization probes for this study. These probes were constructed as chromosome-specific libraries in Bluescribe plasmids and are designated pBS-13, pBS-18, and pBS-21. Elements of these probes bind at numerous sites along the target chromosome and, when detected fluorescently, stain essentially the entire long arm of the target chromosome. The target chromosome number (i.e., the number of chromosomes of the type for which the probe was specific) was correctly determined in 20 of 20 samples in which metaphase spreads were analyzed and in 43 of 43 samples in which interphase nuclei were analyzed; all of these studies were conducted in blind fashion. These results suggest the utility of FISH with composite probes for rapid detection of numerical aberrations in metaphase and interphase amniotic cells.

Aneuploidy↗
PubMed1990

Cloning and expression of the cDNA for a Drosophila insulin-degrading enzyme.

We have previously identified and characterized a metalloproteinase from Drosophila that cleaves insulin and transforming growth factor-alpha, but not epidermal growth factor, at physiological concentrations. On the basis of enzymatic properties and substrate specificity, this enzyme was identified as the Drosophila homolog of the mammalian insulin-degrading enzyme (IDE). We now report the cloning and sequencing of the cDNA coding for the Drosophila IDE (dIDE). Northern blot analysis indicates that the dIDE is translated from a 3.6-kilobase transcript similar in size to one of the two human IDE transcripts. The gene for the dIDE has been mapped to chromosome 3L (77B). The sequence of the dIDE is very similar to that of the human IDE, and both enzymes share limited but significant identity with the bacterial metalloproteinase protease III. Indirect studies based upon inhibitors, degradation products, and microinjected antibodies have suggested that the IDE can initiate cellular insulin degradation in mammalian cells. To determine whether dIDE expressed in mammalian cells can also degrade insulin, we transfected the cDNA into murine NIH3T3 cells. Extracts of the transfected cells showed increased insulin-degrading activity, demonstrating that the dIDE can be functionally expressed in mammalian cells. These results indicate that the properties of the IDE are evolutionarily conserved.

Amino Acid Sequence↗
PubMed1990

Analytical approaches to detection and characterization of disease-linked chromosome aberrations.

Flow karyotyping and FISH with chromosome specific or disease-locus-specific probes are powerful adjuncts to conventional cytogenetic analysis. Flow karyotyping is well suited to quantitative analysis of DNA content changes that occur during structural rearrangement. FISH with probes for repeated sequences allows ready detection of aneuploidy in interphase cells. FISH with whole chromosome composite probes to metaphase spreads facilitates detection of subtle structural changes and allows detection of structural aberrations that occur at frequencies as low as 10(-3). FISH with locus-specific probes facilitates diagnosis of specific genetic diseases, allows phenotype-genotype correlation on a cell-by-cell basis and may be developed into a sensitive method for detection of residual disease.

Chromosome Aberrations↗
PubMed1988

A transcriptionally defective long terminal repeat within an endogenous copy of mouse mammary tumor virus proviral DNA.

Mouse mammary tumor virus proviral DNA is endogenous to most inbred strains of mice but in many strains is not transcriptionally active. This inactivity may be due to defects in the proviruses themselves or to position effects mediated by DNA sequences flanking the proviral units. The transcriptional competence of long terminal repeats (LTRs) derived from endogenous proviral DNA at genetic loci Mtv-8, Mtv-9, and Mtv-17 of the C57BL/6 mouse strain was examined with a transient transfection assay in which gene expression was monitored by expression of chloramphenicol acetyltransferase. LTRs from Mtv-8 and Mtv-9 were able to direct glucocorticoid-induced chloramphenicol acetyltransferase expression in this assay, while the LTR from Mtv-17 was only about 5% as effective. Analysis of chimeric LTRs indicated that the glucocorticoid-inducible transcriptional enhancer element within the Mtv-17 LTR is active when linked to a functional promoter from Mtv-8, whereas the promoter from Mtv-17 is defective in directing hormone-induced gene expression, even when linked to the Mtv-8 glucocorticoid-responsive enhancer. The DNA sequence of transcriptional control regions of the LTRs of all three endogenous proviral units was determined; this analysis revealed that the source of the defect in Mtv-17 is a single G-to-A transition at position-75 with respect to the site of transcription initiation that resides within the previously defined binding site for the transcription factor nuclear factor 1. Competition experiments with a gel electrophoresis mobility shift assay indicated that the affinity of nuclear factor 1 for DNA derived from Mtv-17 is significantly less than for comparable sequences derived from Mtv-8.

Animals↗
PubMed1987

Filter elution analysis of the effect of alpha-difluoromethylornithine on X-ray-induced DNA damage in 9L cells.

We used the filter elution technique to study DNA single- and double-strand scission under denaturing alkaline and nondenaturing conditions in X-irradiated 9L rat brain tumor cells. The amount of DNA damage determined by the alkaline elution assay was similar for different lysis conditions (sodium dodecyl sulfate and sarkosyl) and DNA fluorometric assays (Hoechst 33258 and 3,5-diaminobenzoic acid dyes). Therefore, results of the filter elution assay obtained with the various methods can be compared directly. Using these assays, we found that there was no significant change in the susceptibility to X-ray-induced DNA damage, measured either as single- or double-strand breaks, in 9L cells depleted of polyamines by treatment with alpha-difluoromethylornithine. Results obtained by filter elution are different from results obtained with viscoelastometry, which suggests that the two methods may resolve the effects of changes in DNA structure in different ways.

Animals↗
PubMed1984

Neocarzinostatin-mediated DNA damage and repair in wild-type and repair-deficient Chinese hamster ovary cells.

The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity.

Animals↗