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Biomedical subjects

D I Hoar

Publications and source records attributed to D I Hoar.

At least 37 records · Page 2Linked to original sources
PubMed1989

Genotype and the cryopreservation process affect the levels of aneuploidy and chromosome breakage in cultured human fibroblasts.

Spontaneous micronucleus frequencies were measured in 11 human fibroblast strains, with early-passage cells that had never been frozen and with cells of comparable population doublings that had been cryopreserved in liquid nitrogen. The mean micronucleus frequency of the 11 strains increased from 14.0 +/- 0.7 to 20.4 +/- 1.8/1250 mononucleated cells (P = 0.002) after the freeze-thaw process. The nature of this increase in micronucleus frequency was examined using an immunodetection assay for the in situ identification of kinetochores in micronuclei. The increase in micronucleus frequency occurred primarily in the kinetochore-positive fraction, which is indicative of aneuploidy, but also by an increase in chromosome breakage in several strains. The findings were reproducible in repeat biopsies from two donors. Plating efficiencies of the 11 strains were studied during 1-9 and 10-20 population doublings from primary outgrowth, before freezing and again after freeze-thaw. The mean plating efficiency of frozen-thawed cells before nine doublings was significantly lower than that of cells of similar ages that had never been frozen (P = 0.004). The four strains that had a greater than 25% decrease in plating efficiency post freeze-thaw also had the highest aneuploidy index post freeze-thaw, suggesting that chromosomal imbalance contributes to the observed reduction in growth. We conclude that the genotype and culture manipulations of a fibroblast strain influence the outcome of the micronucleus assay.

Aneuploidy↗
PubMed1989

Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients.

A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to establish a diagnostic assay for the identification of cytomegalovirus (CMV) immediate-early sequences in clinical specimens. Preliminary testing against virus-infected cell cultures indicated that the PCR assay was highly CMV-specific, recognizing both wild-type and laboratory strains of CMV. There was no cross-reactivity with human DNA or with DNA from other herpes viruses. The sensitivity of the assay, using cloned CMV AD169 Eco RI fragment-J as template, was 1 viral genome per 40,000 cells. In a prospective study of CMV infection in bone marrow transplant recipients, the PCR assay correctly identified four patients with confirmed CMV infection. In three of these patients who were followed longitudinally, correlation of DNA reactivity with CMV culture and CMV antibody status over time indicated that DNA was the most sensitive marker for the diagnosis of CMV infection.

Adolescent↗
PubMed1989

Hemopoietic origin of factor XIII A subunits in platelets, monocytes, and plasma. Evidence from bone marrow transplantation studies.

Factor XIII A subunit (FXIIIA) is found in plasma, platelets, and monocytes. The hemopoietic contributions to FXIIIA in these components were studied in patients transplanted with marrows from donors with different FXIIIA phenotypes. In three patients with successful engraftment (by DNA genotyping, red cell phenotyping, and cytogenetic studies) platelet and monocyte FXIIIA changed to donor phenotypes with hematologic recovery. Thus, FXIIIA in platelets and monocytes is synthesized de novo and/or from their progenitor cells. Plasma FXIIIA phenotype change after transplantation was more complex. Patient I changed from phenotype 1-1 (one electrophoretically fast band) to 1-2 (three bands) in 115 d; patients 2 and 3 did not change completely from phenotype 1-2 to 1-1 in up to 458 d, but did show enrichment of the fastest band. Thus, while there is a definite contribution of donor hemopoiesis to plasma FXIIIA, another source of recipient FXIIIA appears to be present to delay or prevent the phenotype change.

Adult↗
PubMed1989

Assessment of the sephadex technique for selection of X-bearing human sperm by analysis of sperm chromosomes, deoxyribonucleic acid and Y-bodies.

The effectiveness of sephadex sperm filtration on altering the sex ratio of human sperm was tested using three different methods. For each ejaculate the ratio of X- and Y-bearing sperm was analysed before and after sephadex filtration using three different methodologies: sperm chromosome analysis after fusion of human sperm with hamster oocytes, deoxyribonucleic acid analysis using the Y-preferential probe pS4 and the fluorescent Y-body test. In 182 sephadex-treated sperm analysed by sperm chromosomes the sex ratio (55.5%X) did not differ significantly from 226 untreated sperm (54.7%X) from the same semen samples. The DNA and Y-body tests demonstrated variability in both degree and pattern of enrichment for replicates but no consistent enrichment for X-chromosome-bearing sperm was observed for any one fraction after sephadex gel filtration.

Autoradiography↗
PubMed1988

Mental retardation, distinct facial changes, short stature, obesity, and hypogonadism: a new X-linked mental retardation syndrome.

We describe a 3-year-old boy and his 2 maternal uncles with moderate to severe mental retardation, short stature, mild obesity, hypogonadism, a low total finger ridge count, and a distinctive face characterized by bitemporal narrowness, almond-shaped palperbral fissures, depressed nasal bridge, anteverted nares, short and inverted-V-shaped upper lip, and macrostomia. Two other males in this family who had similar facial anomalies and developmental delay died in early infancy and midchildhood. This apparently new disorder is reminiscent of, but distinct from, the Prader-Willi syndrome, and is likely inherited as an X-linked recessive trait. Preliminary studies with DNA probes are consistent with an X-linked locus and permit exclusion of distal Xp and Xq regions as the site of this mutation.

Abnormalities, Multiple↗
PubMed1988

Micronucleus assay in human fibroblasts: a measure of spontaneous chromosomal instability and mutagen hypersensitivity.

By comparing fibroblast strains derived from individuals exhibiting chromosome instability and/or mutagen hypersensitivity (Cockayne syndrome, ataxia telangiectasia, and Fanconi anemia) with strains derived from healthy donors, the fibroblast micronucleus assay has been established as a reproducible measure of the genotypic variation in spontaneous or mitomycin C (MMC)-induced chromosomal instability. The patient strains that were moderately or exquisitely sensitive to MMC, whereas the mildly sensitive strain (Cockayne syndrome) overlapped with the control range. The reproducibility of the assay was evaluated within and between experiments. Paired comparison analyses between duplicate cultures and between repeat experiments failed to show any significant differences between micronucleus frequencies within strains, whereas a significant differences in the spontaneous micronucleus frequencies between strains was observed. In addition to its value as a test system for genotoxins, the fibroblast micronucleus assay may be useful for investigating genetically determined hypersensitivity to mutagens, elevated spontaneous chromosomal breakage, and chromosome segregation errors.

Adult↗
PubMed1988

The use of DNA probes to establish parental origin in Down syndrome.

Molecular analysis was performed to determine the parental origin of the extra number 21 chromosome in 20 couples following the birth of a child with standard trisomy 21. The parent of origin was successfully identified in 9 of 20 (45%) using five chromosome-21-specific DNA probes and eight restriction endonucleases by restriction fragment length polymorphism and dosage analysis; seven were of maternal and two of paternal origin. Utilizing the observed allele frequencies, the expected frequencies of informative homozygous matings [2(p2q2)] approximate 10% for seven of eight enzyme/probe combinations; the eighth, TaqI/pPW231F (D21S3), is 3%. The observed phenotype frequencies for all enzyme/probe combinations tested conform closely to predictions by the Hardy-Weinberg law. Strong linkage disequilibrium was observed between the DNA markers of EcoRI and TaqI with probe pPW236B; identical results were obtained with G-95 alpha 1-11a. We were able to demonstrate that although these two probes are of different size, and hence not identical, they detect the same TaqI and EcoRI polymorphisms; therefore both should be assigned to a single locus, D21S11.

DNA↗
PubMed1988

Studies on the mechanism of activation of human natural killer function by interferon and inhibitors of thymidylate synthesis.

Previous publications from this laboratory have demonstrated that agents such as methotrexate (MTX), 5-fluorodeoxyuridine (FUdR), trimethoprim, and D-glucosamine (D-GlcN), which are known to inhibit thymidylate synthesis, can augment human NK activity in vitro. Furthermore, this augmentation was inhibited by exogenous thymidine (TdR) at concentrations of 10(-6) to 10(-7) M. In this report, underlying mechanisms of action of FUdR, D-GlcN, and IFN are compared. Each of these agents increased the lytic activity of effector cells bound to targets but did not increase the percentage of conjugates formed. The augmentation could be induced in a population highly enriched for NK cells (Leu-1 lb positive in phenotype). FUdR and D-GlcN could not induce any augmentation in a Leu-1 lb-negative subpopulation whereas IFN could induce significant lytic activity. alpha-Amanitin, an inhibitor of RNA polymerase II, blocked the activation of NK activity by all three reagents; hence gene expression was required. Comparison of [35S]methionine-labeled proteins by two-dimensional gel electrophoresis revealed that six new proteins were induced in IFN-treated cells. Three of these were similar in pI and molecular weight to the newly synthesized proteins in the D-GlcN-treated cells. One protein was synthesized in increased amounts in the FuDR-treated cells and it was not common to either of the other treatments. Evidence to date is consistent with the hypothesis that separate mechanisms underlie the activation of NK cells by IFN and thymidylate synthesis inhibitors, although the existence of a final common pathway for all NK response modulators cannot be excluded at the present time.

Antibodies, Monoclonal↗
PubMed1988

Kinetochore immunofluorescence in micronuclei: a rapid method for the in situ detection of aneuploidy and chromosome breakage in human fibroblasts.

We have developed a rapid and simple immunodetection assay for the in situ identification of aneuploidy in mitotic fibroblasts. Kinetochore (centromere)-containing micronuclei can be detected easily and rapidly by immunofluorescence. The action of colchicine and its derivatives on the mitotic spindle apparatus of mammalian cells induces chromosome lag and aneuploidy. The treatment of normal human fibroblasts with Colcemid resulted in increased levels of micronuclei. Using an immunofluorescence stain (scleroderma CREST antiserum, biotinylated goat antihuman IgG and streptavidin-Texas Red) to detect the presence of kinetochores, it was observed that 90% of the Colcemid-induced micronuclei contained one or more fluorescent bodies (kinetochores). Cultured skin fibroblasts from a patient with ataxia telangiectasia (AT), which is a chromosome breakage syndrome, were used as a control. The AT fibroblasts exhibited elevated levels of spontaneous micronuclei when compared with normal fibroblasts, and 85% of these micronuclei were kinetochore-negative. This finding supports the hypothesis that the majority of spontaneous micronuclei in AT cells arise from chromosome breakage. The spontaneous micronucleus frequencies for 8 strains of human fibroblasts were in the order of 0.5-2%. Spontaneous levels of kinetochore-positive micronuclei were measured for these 8 strains; in 5 of the strains, about 25% of the micronuclei were kinetochore-positive, and in the other 3 strains approximately 50% of the micronuclei were kinetochore-positive. These data suggest that genetic factors may play a role in the control of the spontaneous levels of chromosome breakage and/or segregation errors which result in aneuploidy.

Aneuploidy↗
PubMed1988

Neurofilament gene expression following beta,beta'-iminodipropionitrile (IDPN) intoxication.

beta,beta'-Iminodipropionitrile (IDPN) is an agent that produces a disorganization of the axonal cytoskeleton with massive accumulation of neurofilaments in the proximal axon. Abnormalities in axonal transport of neurofilament proteins and in their phosphorylation occur in this model. In this study we evaluated the gene expression of neurofilament and other cytoskeletal components at an early, intermediate and late stage of intoxication to determine whether this neuropathy is directly due to or secondarily affects the expression of these components. Specific cytoskeletal mRNA expression was evaluated in the spinal cords of rats treated with IDPN for varying durations using Northern analysis and in situ hybridization. Our results show no qualitative or quantitative alteration in the mRNA expression of the neurofilament triplet, alpha-tubulin, alpha-actin or glial fibrillary acidic protein. We conclude that abnormalities at various stages of cytoskeletal processing such as the early disorganization of the cytoskeleton, the impairment of neurofilament transport, and the long-term redistribution of neurofilaments along the axon are not directly due to, nor do they affect the gene expression of cytoskeletal components in IDPN neuropathy.

Animals↗
PubMed1988

Exaggerated triglyceride accretion in human preadipocyte-murine renal line hybrids composed of cells from massively obese subjects.

To learn about adipose differentiation of precursors from postnatal adipose tissue of lean and massively obese subjects, human omental adipocyte precursor-murine renal adenocarcinoma cell (RAG) hybrids were formed by fusion with polyethylene glycol, and cultured selectively with 50 microM ouabain in hypoxanthine aminopterin thymidine (HAT) medium. Under conditions in which the parent cells did not differentiate, a number of hybrids, which were cloned, revealed morphologic and biochemical evidence of differentiation. In addition to activation of human genes within the common nucleus of the hybrids, murine cytoplasmic activators are probably also involved because heterocaryons (fused cells with two interspecific nuclei) revealed the same phenomenon. Hybrids composed of precursors from massively obese subjects disclosed more frequent and prominent differentiation. Since these hybrids, in contrast to those from the lean, recapitulate this phenomenon in subcultures, they provide the potential system for mapping the human gene(s) responsible for adipose differentiation and its exaggeration in massive obesity.

Adipose Tissue↗
PubMed1987

DNA banking: the effects of storage of blood and isolated DNA on the integrity of DNA.

Long-term storage of DNA is required for a number of genetic studies; prior to extraction, blood samples may be subject to elevated temperatures for variable intervals. We have studied the effect of temperatures ranging from -70 degrees C to +65 degrees C on human blood and on DNA extracted from it. DNA in solution stored at ambient temperatures up to 37 degrees C for 6 months was digestible by three different restriction endonucleases, whereas storage at 45 degrees C is deleterious after 6-7 weeks. DNA can be extracted from blood samples stored at -70 degrees C for at least 2 months or at 23 degrees C for a week or more, but blood stored at these temperatures may yield less high-molecular-weight DNA. Cell pellets from which plasma has been removed also can serve as a source of DNA. Isolated DNA stored dry for years (up to 30) is difficult to dissolve and may appear degraded, but a sample stored dry for 13 years and then in solution at -20 degrees C for 7 years appeared to be intact.

Blood Preservation↗
PubMed1987

Efficacy of commercial condoms in the prevention of hepatitis B virus infection.

Tips of synthetic and natural condoms were filled with serum samples containing either hepatitis B virus, herpes simplex virus, or cytomegalovirus, then fit over an 8-in. mechanical vibrator and inserted vibrating into sterile bath solutions for 30 min. Phosphorus 32-labeled hepatitis B and cytomegalovirus molecular probes and viral culture techniques for herpes simplex and cytomegalovirus were used to determine whether leakage of virus had occurred into the surrounding bath solutions. Natural condoms allowed leakage of hepatitis B virus but not herpes simplex virus or cytomegalovirus, whereas synthetic condoms prevented leakage of all viruses. These results suggest that natural condoms might not be effective in preventing sexually transmitted hepatitis B virus infection.

Contraceptive Devices, Male↗
PubMed1987

Humoral immune response in patients with hemophilia.

Hemophiliacs require frequent infusions of allogeneic proteins to control bleeding. Previous reports have demonstrated that thymus-derived lymphocytes (T cells) from hemophiliacs are antigenically primed to the lyophilized antihemophilic factor and that natural killer cells from hemophiliacs demonstrate impaired response to interferon-beta and -gamma Some aspects of the humoral immune response were investigated in eight patients who require large amounts of Factor VIII. Polyclonal hypergammaglobulinemia was detected in six patients and seven had elevated titers of autoantibodies of various specificities. There was no evidence of impaired concanavalin A-inducible T-suppressor cell activity. Polyclonal immunoglobulin secretion secondary to pokeweed mitogen in vitro was elevated in three of eight patients and depressed in five. Spontaneous production of both B-cell growth and differentiation factors (BCGF and BCDF) was elevated but mitogen-induced production was impaired. These data demonstrate that the humoral immune response of hemophiliacs may be chronically stimulated, thus impairing their ability to respond to new antigens such as viruses.

AIDS-Related Complex↗
PubMed1987

Hemophilia B (Christmas disease) variants and carrier detection analyzed by DNA probes.

We have used two strategies to study 14 hemophilia B families from 11 kindreds for possible carrier detection and prenatal diagnosis. First, we sequentially used the Factor IX probes (sequentially with restriction enzymes Taq I, Xmn I, and Dde I), and the linked probes p45h (Taq I), p45d (Pst I), and 52a (Taq I) for restriction fragment length polymorphism (RFLP) analysis. Second, we searched for useful variant Taq I digestion fragments using the Factor IX complementary DNA. Two separate new Taq I variants in exon VIII were identified. Using both strategies, 11 of 14 families (from 9 of 11 kindreds) were informative for further studies. In five kindreds studied in detail, the carrier status of all 11 at risk females was determined and prenatal diagnosis could be offered to the offsprings of each of the six carriers identified. Thus, in this study, we have identified a higher proportion of informative families than has previously been reported.

DNA↗
PubMed1986

Molecular detection of a translocation (Y;15) in a 45,X male.

A 45,X male individual was shown to have a translocation of Y-chromosome material to the short arm or proximal long arm of chromosome 15. This translocation was detected by genomic DNA blotting and in situ hybridization with Y-chromosome-specific DNA probes.

Chromosome Banding↗