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Biomedical subjects

D Pinkel

Publications and source records attributed to D Pinkel.

At least 127 records · Page 7Linked to original sources
PubMed1989

Rapid human chromosome aberration analysis using fluorescence in situ hybridization.

We have used in situ hybridization of repeat-sequence DNA probes, specific to the paracentromric locus 1q12 and the telomeric locus 1p36, to fluorescently stain regions that flank human chromosome 1p. This procedure was used for fast detection of structural aberrations involving human chromosome 1p in two separate experiments. In one, human lymphocytes were irradiated with 0, 0.8, 1.6, 2.4 and 3.2 Gy of 137Cs gamma-rays. In the other, human lymphocytes were irradiated with 0, 0.09, 0.18, 2.0, 3.1 and 4.1 Gy of 60Co gamma-rays. The frequencies (per cell) of translocations and dicentrics with one breakpoint in 1p and one elsewhere in the genome were determined for cells irradiated at each dose point. These frequencies both increased with dose, D, in a linear-quadratic manner. The delta, alpha, and beta coefficients resulting from a fit of the equation f(D)=delta + alphaD + betaD2 to the translocation frequency dose-response data were 0.0025, 0.0027 and 0.0037 for 137Cs gamma-rays, and 0.0010, 0.0041, and 0.0057 for 60Co gamma-rays. The delta, alpha, and beta coefficients resulting from a fit to the dicentric frequency dose-response data were 0.0005, 0.0010 and 0.0028 for 137Cs gamma-rays and 0.0001, 0.0002 and 0.0035, for 60Co gamma-rays. Approximately 32,000 metaphase spreads were scored in this study. The average analysis rate was over two metaphase spreads per minute. However, an experienced analyst was able to find and score one metaphase spread every 10s. The importance of this new cytogenetic analysis technique for biological dosimetry and in vivo risk assessment is discussed.

Cesium Radioisotopes↗
PubMed1989

Three-dimensional analysis of the organization of human chromosome domains in human and human-hamster hybrid interphase nuclei.

This report describes the intranuclear organization of chromosomes in human-hamster hybrid nuclei and in human cell nuclei. The target chromosomes were stained using in situ hybridization with biotinylated, chromosome-specific DNA probes. Bound probe was detected with fluorescein-avidin. Hybridizations were performed to fixed nuclei in aqueous suspension in order to preserve their three-dimensional morphology. Total nuclear DNA was stained with DAPI. Three-dimensional information about the organization of DNA and probe within the nucleus was obtained by optical sectioning. The human chromosomes in human-hamster hybrid nuclei were found to be confined to 'domains' that were maintained during the cell cycle. Different spatial localization patterns of the human chromosomes were seen in interphase nuclei of two different hybrid cell lines. The positions of chromosome-specific repetitive sequences in human fibroblast interphase nuclei were also studied using probes for the telomeric region of chromosome 1p (1p36), the centromeric region of chromosome 9 (9q12) and the long arm of the Y chromosome (Yq12). These studies showed that the two 1p telomeric loci are located near the nuclear surface. The chromosome 9 centromeric loci are similarly located. Simultaneous hybridization of the chromosome 1 telomeric probe (target size approximately 200 kb; b, base) and the Y-specific probe (target size greater than 2Mb), demonstrate that the binding sites of the two probes can be distinguished in the same nucleus on the basis of domain size.

Animals↗
PubMed1988

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization.

Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluorescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluorescence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase in consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1988

Enzymatic production of single-stranded DNA as a target for fluorescence in situ hybridization.

This study demonstrates that Exonuclease III (Exo III) can be used to produce sufficient single-stranded (ss)DNA in chromosomes and cells to allow in situ hybridization. In this study, all of the probes were modified with biotin and the probe binding was visualized with fluorescein-labeled avidin. Exo III digestion starting at naturally occurring breaks in methanol-acetic acid preparations produced enough ssDNA for strong hybridization when human genomic DNA was used to probe human chromosomes. Pretreatment with the endonucleases EcoRI, Hind III and BamHI was used to produce more sites for initiation of Exo III digestion when using a chromosome-specific repetitive probe specific to a small chromosomal subregion near the telomere of human chromosome 1(1p36). The fluorescence intensity following hybridization to Exo III-treated targets was roughly equal to that following hybridization to thermally denatured targets, but background fluorescence was lower.

Animals↗
PubMed1988

Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4.

Chromosomes can be specifically stained in metaphase spreads and interphase nuclei by in situ hybridization with entire chromosome-specific DNA libraries. Unlabeled human genomic DNA is used to inhibit the hybridization of sequences in the library that bind to multiple chromosomes. The target chromosome can be made at least 20 times brighter per unit length than the others. Trisomy 21 and translocations involving chromosome 4 can be detected in metaphase spreads and interphase nuclei by using this technique.

Cells, Cultured↗
PubMed1987

Cytosine arabinoside/cyclophosphamide pulses during continuation therapy for childhood acute lymphoblastic leukemia. Potential selective effect in T-cell leukemia.

One hundred seventy-seven children with acute lymphoblastic leukemia (ALL) were admitted to a study designed to determine whether pulses of cytosine arabinoside (ara-C) and cyclophosphamide (cyclo) would improve disease-free survival (DFS). All patients received vincristine, prednisone, and asparaginase for remission induction, CNS prophylaxis with cranial irradiation and intrathecal methotrexate, and continuation therapy with 6-mercaptopurine plus methotrexate. Forty-seven of 101 patients with non-T ALL and 18 of 26 patients with T-cell ALL received ara-C/cyclo pulses every eight weeks during continuation therapy. The age, sex, and initial white cell count distributions were similar in both treatment groups. Patients with non-T-cell ALL had similar DFS with or without ara-C/cyclo pulses (36% versus 48%; P = 0.32). Ara-C/cyclo pulses significantly improved DFS in children with T-cell ALL (36% versus 0%; P = 0.015). Toxicities of the ara-C/cyclo pulses included reversible pancytopenia, drug induced fever, fever associated with neutropenia, and death in one patient from systemic candidiasis while neutropenic. This is the first clinical evidence to indicate that the combination of ara-C/cyclo used during continuation therapy is selectively beneficial in T-cell ALL.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1987

Flow sorting of X and Y chromosome-bearing spermatozoa into two populations.

The only established difference on which to base the separation of X and Y chromosome-bearing spermatozoa is chromosomal constitution. This difference is quantifiable both from chromosome morphology (karyotype) and from DNA content. Flow cytometric techniques were used to measure relative DNA content of the X and Y populations and to flow-sort spermatozoa from Chinchilla laniger. Epididymal spermatozoa were recovered in PBS, fixed in 80% ethanol, treated with papain and dithioerythritol, and stained for DNA with Hoechst 33342. Sperm nuclei were analyzed and sorted on an EPICS V flow cytometer/cell sorter, modified specifically for spermatozoa. Two clearly resolved peaks (coefficient of variation less than 1.5%) with approximately 7.5% difference in DNA content between X and Y chromosome-bearing spermatozoa were evident. Sperm nuclei were sorted from a portion of the X and Y peaks at a rate of 55 nuclei/sec for each population. Purities of individual X and Y populations averaged 95% as determined by reanalysis of the sorted populations. Successful sorting of Chinchilla X and Y chromosome-bearing spermatozoa into separate populations may aid in the identification of a biochemical marker that could be used to discriminate between the two sperm populations and lead to a practical procedure for sexing spermatozoa.

Animals↗
PubMed1987

Incidence of chromosome aberrations in mammalian sperm stained with Hoechst 33342 and UV-laser irradiated during flow sorting.

The separation of two sperm populations is possible using the technique of flow sorting, provided that a significant difference exists in the DNA content of X- and Y-bearing sperm. In order to ascertain whether or not chromosome damage was induced in sorted sperm, chromosome preparations were made from isolated sperm that had been microinjected into hamster eggs. While egg chromosomes exhibited a low frequency of chromosome aberrations, ranging from 4 to 7%, a large proportion of sperm cells exhibited chromosome damage. Between 29% of unstained and unsorted sperm and 38% of stained and unsorted sperm exhibited some type of chromosomal abnormality and this proportion increased to 50% in sorted sperm. If only damaged sperm nuclei are considered, the two unsorted sperm groups had a mean of 0.6 breaks, 0.8 triradial exchanges, and 0.2 quadriradial exchanges per nucleus. However, sorted sperm, which were stained with a fluorochrome and exposed to UV-laser irradiation, exhibited a mean of 2.9 breaks, 2.6 triradial, and 1.9 quadriradial exchanges per nucleus in which damage occurred. These observations indicate that the treatments and manipulations to which sperm nuclei are subjected during flow sorting cause chromosomal aberrations, and that exposure of the cells to UV-laser irradiation contributes substantially to the chromosome damage observed.

Animals↗
PubMed1987

Chromosome-specific alpha satellite DNA from human chromosome 1: hierarchical structure and genomic organization of a polymorphic domain spanning several hundred kilobase pairs of centromeric DNA.

The human alpha satellite repetitive DNA family is organized as distinct chromosome-specific subsets localized to the centromeric region of each chromosome. Here, we report he isolation and characterization of cloned repeat units which define a hierarchical subset of alpha satellite on human chromosome 1. This subset is characterized by a 1.9-kb higher-order repeat unit which consists of 11 tandem approximately 171-bp alpha satellite monomer repeat units. The higher-order repeat unit is itself tandemly repeated, present in at least 100 copies at the centromeric region of chromosome 1. Using pulsed-field gel electrophoresis we estimate the total array length of these tandem sequences at the centromere of chromosome 1 to be several hundred kilobase pairs. Under conditions of high stringency, the higher-order repeat probe hybridizes specifically to chromosome 1 and can be used to detect several associated restriction fragment length DNA polymorphisms. As such, this probe may be useful for molecular and genetic analyses of the centromeric region of human chromosome 1.

Chromosome Mapping↗
PubMed1986

Modification of a laser-based flow cytometer for high-resolution DNA analysis of mammalian spermatozoa.

Modification of a Coulter EPICS V orthogonal laser-based flow cytometer/cell sorter allows resolution of X and Y mammalian sperm populations based on DNA content. The modification consists of beveling the sample injection tube situated in the flow chamber, adding a second fluorescence detector directly forward along the laser beam axis, and routing the collected fluorescence through an optical fiber bundle to one of the existing photomultiplier tubes. The X and Y chromosome-bearing spermatozoa from the bull, boar, and ram can be resolved using this system.

Animals↗
PubMed1986

Orientation measurements of microsphere doublets and metaphase chromosomes in flow.

Obtaining information about the shape of particles from slit-scan profiles is facilitated if the particles are oriented. Elongated particles orient in the nozzle of flow cytometers, but orientation may be disrupted before the particles get to the point of measurement. We have used our slit-scan flow cytometer to investigate the orientation of microsphere doublets in a liquid jet in air, in flow across a glass surface, and in a 200-microns-square capillary tube as a function of distance from the flow chamber nozzle. Particles were classified as being oriented if there was a centrally located dip in the slit-scan profile. Essentially all the doublets in the jet were oriented, and no disorientation was noted over the distances measured (up to 10 mm from the nozzle). Particle orientation was maintained for 80 microns in flow across a glass surface. In the capillary-type flow chamber, essentially all of the particles were oriented at the tube entrance and for several millimeters into the tube. There then occurred a region where particle tumbling started and progressively fewer doublets met the orientation criteria. The distance to where tumbling began could be estimated by calculating the length required to establish the parabolic flow profile in the tube. Finally, the fraction of oriented particles reached a constant value that did not change with increased distance into the tube. When sample was injected off axis (i.e., halfway between the chamber center and the chamber walls), particle tumbling began closer to the tube entrance.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1986

Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization.

This report describes the use of fluorescence in situ hybridization for chromosome classification and detection of chromosome aberrations. Biotin-labeled DNA was hybridized to target chromosomes and subsequently rendered fluorescent by successive treatments with fluorescein-labeled avidin and biotinylated anti-avidin antibody. Human chromosomes in human-hamster hybrid cell lines were intensely and uniformly stained in metaphase spreads and interphase nuclei when human genomic DNA was used as a probe. Interspecies translocations were detected easily at metaphase. The human-specific fluorescence intensity from cell nuclei and chromosomes was proportional to the amount of target human DNA. Human Y chromosomes were fluorescently stained in metaphase and interphase nuclei by using a 0.8-kilobase DNA probe specific for the Y chromosome. Cells from males were 40 times brighter than those from females. Both Y chromosomal domains were visible in most interphase nuclei of XYY amniocytes. Human 28S ribosomal RNA genes on metaphase chromosomes were distinctly stained by using a 1.5-kilobase DNA probe.

Aneuploidy↗
PubMed1986

Assessment of spermatozoal function using dual fluorescent staining and flow cytometric analyses.

Spermatozoa from bulls, boars, dogs, horses, mice, and men were examined using a fluorogenic stain consisting of the membrane-permeant substrate carboxyfluorescin diacetate (CFDA) and the relatively membrane-impermeant nuclear stain propidium iodide (PI). Three distinct populations of spermatozoa were discernible in samples from each species upon microscopic examination. Individual spermatozoa, presumed to be viable because of their motility, retained products of the fluorescein chromophore throughout the cell. A second population of spermatozoa in which the nuclei stained red with PI retained the green fluorescein fluorophore mainly in the acrosome. A third population, presumed to be degenerate spermatozoa, possessed only red fluorescent nuclei. These populations were quantified using dual parameter flow cytometry in 14 samples of cryopreserved bovine spermatozoa for which fertility and seminal quality data were available. Flow cytometric analyses were highly correlated with other seminal quality measurements. Sequential flow cytometric analyses provided the ability to rapidly quantitate changes in specific fluorescently stained populations. The ability to make rapid quantitative measurements should allow development of new and presumably more reliable information on the functional aspects of spermatozoa.

Acrosome↗