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Biomedical subjects

S A Latt

Publications and source records attributed to S A Latt.

At least 109 records · Page 6Linked to original sources
PubMed1979

Sister chromatid exchange induction resulting from systemic, topical, and systemic-topical presentations of carcinogens.

Chinese hamster cheek pouch mucosal cells were examined for in vivo sister chromatid exchange formation resulting from the exposure of animals to carcinogens presented in three manners: systemic, topical, and systemic-topical combination. The systemic presentation of cyclophosphamide (5 or 10 mg/kg) through i.p. injection resulted in an increase in sister chromatid exchanges from 4.8 to 9.9 per cell. Topical application of 7,12-dimethylbenz(a)anthracene (0.5% in mineral oil, 0.1 ml) resulted in an increase in the sister chromatid exchange frequency to 11.5/cell, as compared with a value of 5.0/cell in animals treated only with mineral oil. Systemic administration of 8-methoxypsoralen (0.5, 1, 2.5, or 5 mg/kg) through i.p. injection, followed by activation with topical near-ultraviolet light (3.75 x 10(4) ergs/sq mm at 365 nm) resulted in an increase in sister chromatid exchange, reaching 15.4/cell at 5 mg 8-methoxypsoralen per kg. Exposure of animals to 8-methoxypsoralen or near-ultraviolet light alone, but not in combination, did not produce an increase in sister chromatid exchange. Sister chromatid exchange frequencies in cheek pouch cells were also compared with sister chromatid exchange frequencies in marrow cells of identically treated animals to assess the importance of exposure mode and tissue specificity in sister chromatid exchange formation.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1978

Analysis of sister chromatid exchange and chromosome replication kinetics using Brd-U-dye techniques.

The basis of BrdU-dye techniques for detecting DNA synthesis in cytological preparations is reviewed. General procedural modifications are described for applying this approach for the study of chromosome replication kinetics and sister chromatid exchange formation. Implications of sister chromatid exchange analysis for detecting mutagen-carcinogens and for differentiating human chromosome fragility diseases predisposing to neoplasia are discussed.

Animals↗
PubMed1978

Bromodeoxyuridine tablet methodology for in vivo studies of DNA synthesis.

5-Bromodeoxyuridine (BrdU) tablets with different physical characteristics are useful in a wide variety of studies requiring detection of DNA replication in vivo. These tablets can effect a high substitution of BrdU in DNA, thereby permitting sister chromatid differentiation in chromosomes stained with 33258 Hoechst alone or in conjunction with Giemsa. Baseline and cyclophosphamide-induced in vivo sister chromatid exchange frequencies in mouse spleen, marrow, and thymus were measured and found to be significantly greater than those in spermatogonia. Sister chromatid exchange analysis was also extended to mouse liver and to Chinese hamster and Armenian hamster marrow cells. Sister chromatid differentiation was observed in Armenian hamster meiotic tissue, and evidence for interhomolog chromatid exchange obtained.

Animals↗
PubMed1978

Enhancement of banding patterns in human metaphase chromosomes by energy transfer.

Intermolecular energy transfer between appropriately chosen pairs of dyes can be used to induce or enhance banding patterns in human metaphase chromosomes. Energy transfer, calibrated by fluorometric studies on soluble dye.DNA complexes, can also be detected by photometric measurements on cytological preparations of metaphase chromosomes stained with pairs of fluorochromes. If a fluorescent dye with one type of binding or quantum yield specificity (e.g., quinacrine, 33258 Hoechst, or chromomycin A(3)) is employed together with a counterstain (e.g., actinomycin D, 7-aminoactinomycin D, or methyl green) exhibiting a complementary base pair binding specificity and satisfying spectral overlap criteria for energy transfer, contrast in fluorescence from the first dye is enhanced in specific subsets of standard chromosome bands. Extensive energy transfer presumably suppress donor fluorescence except in chromosomal region containing clusters of at least 20 base pairs predominantly of one type, within which the donor but not the acceptor can bind and fluoresce. Quinacrine-bright polymorphic regions are especially resistant to fluorescence quenching by counterstains with G.C binding specificity, strengthening the evidence that these latter regions are highly enriched for A.T base pair clusters. The ability to highlight selectively many such polymorphic regions may prove of further, practical, utility in a number of cytogenetic problems.

Chromosomes, Human↗
PubMed1978

Search for DNA interchange corresponding to sister chromatid exchanges in Chinese hamster ovary cells.

Chinese hamster ovary cells (CHO) grown for one cycle in bromodeoxyuridine (BrdU) contain a small amount (0.5%) of unusually dense double stranded DNA. This dense DNA has been previously interpreted as being bifilarly substituted with BrdU and hence evidence that sister chromatid exchange (SCE) formation proceeds via the Holliday model of recombination. However, the amount of this dense DNA is 100 times greater than that expected based on the SCE frequency in similarly cultured CHO cells, and it is not increased by treating the cells with mitomycin C. Moreover, contrary to expectations for bifilary substituted DNA, the amount of this dense DNA is not reduced by growing BrdU-labeled cells for a second cycle in TdR. Finally, DNA isolated from CHO cells contains a minor band (0.5%) with a density 0.025 gm/cc greater than that of the main band, whether or not BrdU has been incorporated. These results call into question the identification of this unusually dense DNA as bifilarly substituted and hence its previously postulated relationship to SCE formation.

Bromodeoxyuridine↗
PubMed1978

Multiple cytogenetic methods used to identify a new structural rearrangement of the human X chromosome.

A woman with primary amenorrhea and pure gonadal dysgenesis had two cytogenetically abnormal cell lines. The karyotype was 45,X in 56--95% of mitosis from lymphocytes and skin fibroblasts. In the remaining 5--44% of the cells there was, in addition to a normal X, a structurally abnormal X chromosome interpretable as pter leads to q21::q11 leads to pter or pter leads to q21::q13 leads to pter. The abnormal X chromosome was heterocyclic and had a normal centromere plus an extra C band in the long arm. Detailed interpretation of the structural rearrangements of this chromosome required the use of both Q-, G-, and C-banding and the BrdU-Hoechst 33258 technique.

Adult↗
PubMed1978

Characterization of sister chromatid exchange induction by 8-methoxypsoralen plus near UV light.

The combination of 8-methoxypsoralen and near UV light is highly effective in inducing sister chromatid exchanges (SCEs) in Chinese hamster ovary cells. Appreciable increases in SCEs can be effected by treatments compatible with cell survival, and effects of a single dose of alkylation persist over multiple generations. Both the frequency and location of SCEs induced at different times within the DNA synthesis period varies in a manner indicating that exchange induction is restricted to regions which replicated during or after DNA damage.

Animals↗
PubMed1977

X-chromosome replication in parthenogenic benign ovarian teratomas.

Somatic cells from human females undergo X-differentiation, which curtails expression of most, if not all, of the genes on one X-chromosome. According to the Lyon hypothesis, the designation of whick X will be inactive in eutherian females is random. However, in spite of the obvious biologic importance of X chromosome differentiation, little is known about either the mechanism of this process or the role played by fertilization. Benign ovarian teratomas provide a system for assessing the importance of fertilization in X chromosome differentiation. Biochemical and cytologic data indicate that these teratomas are of germ-cell origin. They are comprised entirely of one of the products of the first meiotic division and are thus parthenogens. In the absence of appropriate recombinational events, ovarian teratomas are consistently homozygous at autosomal loci, even when the host is heterozygous. Analysis of X chromosome replication kinetics provides one additional approach for investigating X-differentiation in individual teratoma cells. We utilized BrdU-dye techniques to study terminal replication patterns in ovarian teratomas and in normal fibroblasts and peripheral lymphocytes from the same individuals. The results confirm that human ovarian teratomas possess a single late-replicating X chromosome. Moreover, the pattern of replication in this X is identical to that in normal fibroblasts, but different from that usually observed in peripheral lymphocytes. Thus, if late replication is an accurate gauge of X-inactivation, the data confirm that X-inactivation can occur without fertilization.

Cell Differentiation↗
PubMed1977

Fluorescent probes of chromosome structure and replication.

Procedures employing fluorescent dyes or Giemsa stain have been utilized to differentiate methaphase chromosomes into longitudinal segments termed bands. In spite of the immense practical utility of chromosome banding, the chemical basis of banding patterns remains incompletely understood. Physical chemical studies have elucidated the modes and specificities of the interaction of fluorescent dyes such as quinacrine, 33258 Hoechst, daunomycin, chromomycin A3 and 7-aminoactinomycin D with DNA and chromatin. However, it is not clear that all aspects of chromosome staining are explainable in terms of the optical properties of soluble dye-DNA complexes. BrdU-dye techniques in which chromosome staining depends on the schedule of BrdU incorporation by cells, have been used for cytological studies of chromosome structure and replication. These procedures have revealed a close association between quinacrine or Giemsa bands and late replicating chromosomal regions. Biochemical studies on chromatin differentially labelled according to replication timing may thus prove useful for investigating the molecular basis of chromosome banding.

Animals↗
PubMed1977

A simplified technique for in vivo analysis of sister-chromatid exchanges using 5-bromodeoxyuridine tablets.

A small tablet of 5-bromodeoxyuridine (BrdU), implanted subcutaneously in a mouse, provides sustained release of base analog sufficient to effect substitution of DNA throughout an entire replication period. As illustrated by studies of mouse bone-marrow and spleen cells in the presence or absence of cyclophosphamide, this depot method of BrdU administration greatly simplifies in vivo analysis of sister-chromatid-exchange formation.

Animals↗
PubMed1977

Automatic measurement of sister chromatid exchange frequency.

An automatic system for detecting and counting sister chromatid exchanges in human chromosomes has been developed. Metaphase chromosomes from lymphocytes which had incorporated 5-bromodeoxyuridine for two replication cycles were treated with the dye 33258 Hoechst and photodegraded so that the sister chromatids exhibited differential Giemsa staining. A computer-controlled television-microscope system was used to acquire digitized metaphase spread images by direct scanning of microscope slides. Individual objects in the images were identified by a thresholding procedure. The probability that each object was a single, separate chromosome was estimated from size and shape measurements. An analysis of the spatial relationships of the dark-chromatid regions of each object yielded a set of possible exchange locations and estimated probabilities that such locations corresponded to sister chromatid exchanges. A normalized estimate of the sister chromatid exchange frequency was obtained by summing the joint probabilities that a location contained an exchange within a single, separate chromosome over the set of chromosomes from one or more cells and dividing by the expected value of the total chromosome area analyzed. Comparison with manual scoring of exchanges showed satisfactory agreement up to levels of approximately 30 sister chromatid exchanges/cell, or slightly more than twice control levels. The processing time for this automated sister chromatid exchange detection system was comparable to that of manual scoring.

Autoanalysis↗
PubMed1977

Fluorometric detection of deoxyribonucleic acid synthesis; possibilities for interfacing bromodeoxyuridine dye techniques with flow fluorometry.

Fluorometric detection of the biosynthetic incorporation of 5-bromodeoxyuridine (BrdU) into deoxyribonucleic acid has permitted cytologic studies of chromosome structure, replication, and repair. Some of these phenomena, previously detected using BrdU-dye techniques on fixed microscopic preparations, should be particularly amenable to analogous experimentation in fluorescence flow systems. Problems involved in interfacing BrdU-dye methodology with flow fluorometry are discussed. The effects of certain chemical modifications of bisbenzimidazole dyes on their spectroscopic properties and potential use for detecting BrdU incorporation into unfixed cells are described. Data on the use and energy transfer characteristics of a pair of deoxyribonucleic acid-binding dyes (33258 Hoechst and ethidium bromide) capable of simultaneously providing information about BrdU substitution and total deoxyribonucleic acid content are presented.

Bisbenzimidazole↗
PubMed1977

Flow cytometric analysis of bromodeoxyuridine-substituted cells stained with 33258 Hoechst.

This paper describes a flow-cytometric application of the quenching of fluorescence from 33258 Hoechst stained Chinese hamster ovary-line cells due to the incorporation of 5-bromo-deoxyuridine (BrdU) into the cellular deoxyribonucleic acid. Cells were grown for 24 hr in medium containing BrdU in concentrations ranging from 1 x 10(-8) to 1 x 10(-4) M. For each concentration we measured the average fluorescence as determined by flow cytometry, the extent of BrdU substitution and the effect of the BrdU on cell growth. We determined that a BrdU concentration of 1 x 10(-5) M resulted in sufficient substitution to quench the fluorescence from 33258 Hoechst by a factor of 4, allowing discrimination between cycling and noncycling cells. The extent of BrdU substitution after growth for 24 hr in this concentration of BrdU was 64%. These data indicate the feasibility of detecting deoxyribonucleic acid synthesis in whole cells using the 33258 Hoechst-BrdU methodology.

Benzimidazoles↗