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Biomedical subjects

G M Martin

Publications and source records attributed to G M Martin.

At least 163 records · Page 9Linked to original sources
PubMed1983

Fragile X expression studied by clonal analysis and somatic cell hybridization.

The expression of the fragile site on the X chromosome was examined in euploid somatic cell hybrids to determine if a normal genome could complement the abnormal genome and suppress fragile X expression. Fibroblasts from the patient showed the fragile X in 8% to 12% of cells, and fibroblasts from the normal individual showed no fragile X expression. Six fibroblast clones from the patient showed a variable frequency of fragile X expression (from 6.6% to 12%), suggesting that the fragile X is not expressed in a clonal fashion. Three clones from the normal individual did not show the fragile X. Four hybrid clones showed the fragile X in 5.6%, 4.0%, 7.0%, and 4.0% of cells, respectively, indicating that fragile X expression is not completely suppressed by the normal genome.

Cells, Cultured↗
PubMed1983

Comparative rates of decline in the primary cloning efficiencies of smooth muscle cells from the aging thoracic aorta of two murine species of contrasting maximum life span potentials.

Primary cloning assays of thoracic aortic smooth muscle cells from an F1 hybrid strain of Mus musculus demonstrated linear regressions of replicative potentials as functions of donor age (6-30 months), with regression coefficients, in two independent cohorts, of -1.69 +/- 0.27 (SE) and -1.88 +/- 0.19 (SE) clones per milligram wet weight of intima-media per month and correlation coefficients of -0.83 and -0.92 (P less than 0.001). Secondary cloning (dilute plating from first passages) also demonstrated a high negative correlation (r = -0.90) between donor age and cloning efficiency, thus implicating intrinsic differences in cell populations. Comparable primary cloning assays on the aortas of aging cohorts of Peromyscus leucopus, a murine species with a maximum life span potential approximately twice that of Mus musculus, yielded about twice the number of clonable smooth muscle cells per unit weight; the rate of decline with age was slightly but significantly greater (P less than 0.01). Electron-microscopic studies revealed cellular alterations confined to the first subintimal layer of aortas from mice (Mus musculus) 18 months and older.

Age Factors↗
PubMed1982

Evidence that a critical threshold of DNA polymerase-alpha activity may be required for the initiation of DNA synthesis in mammalian cell heterokaryons.

The specific activity of DNA polymerase (90% alpha) was determined in nine "neoplastoid" cell lines (Martin and Sprague, 1973) and in three different strains of HDF (human diploid fibroblast-like cells), all examined in logarithmic phases of growth. This was compared to the ability of each cell type to "rescue" (reinitiate DNA synthesis in) senescent HDF cells subsequent to polyethylene glycol-mediated cell fusions. A sharp "threshold" value of DNA polymerase activity was observed below which reinitiation of DNA synthesis in heterokaryons with senescent HDF does not occur. This threshold was especially obvious when the specific activity of DNA polymerase (p moles dTTP incorporated per mg protein or per cell) was divided by the percent of S-phase cells present in each culture as determined by flow microfluorometry. Our results indicate that the specific activity of DNA polymerase-alpha (or some other factor tightly coregulated with it) in "recessive" cell types (those unable to rescue senescent cells) is only about two times this theoretical "threshold" value, and that fusion of recessive cell types to senescent HDF cells reduces the specific activity in the heterokaryon to below this minimum, thus preventing the cells from entering S phase.

Cell Cycle↗
PubMed1982

Cultivated cells from mid-trimester amniotic fluids: IV. Cell type indentification via one and two-dimensional electrophoresis of clonal whole cell homogenates.

Clones of cultivated amniotic fluid cells that have distinct morphologic and growth characteristics (F, AF and E-type) were examined by one-dimensional SDS polyacrylamide gel electrophoresis (SDS-PAGE) and by two-dimensional electrophoresis employing isoelectric focusing and SDS-PAGE (IEF-PAGE). No qualitative differences in band pattern were observed in SDS-PAGE between the various amniotic fluid cell types, but consistent quantitative differences in the ratios of four bands of presumed filamentous proteins provided good distinction between amniotic fluid cells and postnatal skin fibroblast-like cells. By adding separation on the basis of electrical charge to that of molecular size (IEF-PAGE), we observed reproducible qualitative differences in the protein spot patterns between F and both AF and E-type amniotic fluid cells. At least eight discrete proteins appear not be synthesized by prenatal F-type cells in comparison with their isogenic AF and E counterparts under identical culture conditions. The two-dimensional electrophoretic patterns thus confirm that F and AF amniotic cells, in spite of their morphologic and growth kinetic similarities, are developmentally distinct cell types that retain their differentiated states in culture.

Amniotic Fluid↗
PubMed1982

Interphase flow-cytogenetics: correlation of DNA fluorescence to aneuploidy in human fibroblast cultures.

Improvements in the accuracy of DNA content determination by flow cytometry might allow the detection of aneuploidy in interphase cells to serve as an adjunct to conventional cytogenetics. We compared alternate methods of staining and fluorescence standardization in analyses of human diploid and constitutionally aneuploid cultured fibroblast-like cells. Optimum results were obtained using the dye DAPI (4,6-diamidino-2-phenylindole) and an avian red blood cell standard. The standard error of the estimate of DNA content by this technique was 0.7%-0.96%, approaching more closely than previously the range necessary for reliable discrimination of numerical chromosomal aberrations.

Aneuploidy↗
PubMed1982

Encephalomyocarditis virus as a probe of errors in macromolecular synthesis in aging mice.

A viral probe was used in attempts to develop an in vivo test of the hypothesis that cellular senescence is accompanied by an increased rate of errors in macromolecular synthesis. Young and senescent Balb/cNNia mice were infected with encephalomyocarditis (EMC) virus. No differences in pattern of infection or titers of virus in brain and heart were observed between the two age groups. The yield of virus in control experiments was shown to be reduced by growth in the presence of 5-fluorouracil or a mixture of three amino acid analogs. Since the growth of this virus is highly dependent upon host cell synthetic machinery, these results are thought to suggest that substantial elevations in the rate of errors in macromolecular synthesis in these tissues do not occur with age. Further studies might allow a more precise determination of whether there is an age correlation of in vivo error rates; for the EMC virus, a selectable marker suitable for the quantitation of rates of mutation in vivo has so far not been obtainable.

Aging↗
PubMed1982

Syndromes of accelerated aging.

A systematic review of the more than 2,000 genetic loci of man cataloged by McKusick indicated that approximately 7% may play a role in modulating the rates of development of various aspects of the senescent phenotype. Assuming an upper limit of about 100,000 loci in man, numerous alleles at approximately 7,000 loci could be contributing to characteristic patterns of aging in individual human beings. Point mutations or chromosomal aberrations involving such loci may result in various progeroid syndromes. These have been classified into two categories: segmental progeroid syndromes, which involve multiple aspects of the senescent phenotype, and unimodal progeroid syndromes, in which predominantly one aspect of the phenotype is involved. Two different examples of segmental progeroid syndromes were discussed: the Werner syndrome (an autosomal recessive) and the Down syndrome (trisomy 21). Examples of unimodal progeroid syndromes included familial hypercholesterolemia (accelerated atherogenesis), xeroderma pigmentosum (acceleration of skin aging, including age-related neoplasms), and certain forms of intestinal polyposis (acceleration of adenocarcinoma of the colon). It is remarkable and encouraging that the biochemical genetic basis of many progeroid syndromes, including all of those mentioned above, may be amenable to investigation with cultured mesenchymal somatic cells from individual subjects. For example, cells from patients with the Werner's syndrome have a striking limitation of their in vitro replicative life-spans and undergo extensive chromosomal rearrangements. These abnormalities are presumably related to an enzyme deficiency which, in principle, could be identified by biochemical studies of cultured cells.

Alleles↗
PubMed1981

Preparation of cell-size unilamellar liposomes with high captured volume and defined size distribution.

Cell-size unilamellar vesicles were made by removing solvents from microscopic chloroform spherules containing smaller water droplets within. The average diameter of the vesicles in a typical preparation was 9.2 mum, comparable to that of human erythrocytes (7 mum). The standard deviation of the size distribution was 3.0 mum. The unilamellarity and bilayer unit membrane of vesicles were demonstrated by transmission electron microscopy. Materials so far successfully incorporated into vesicles include glucose, sucrose, Arsenazo III and Ponceau S dyes, thymidine triphosphate, methotrexate, agarose, collagen, ferritin, polyadenylic aid, DNA, and whole bacteria. The captured volume per milligram of lipids (up to 144 microliter/mg) was almost an order of magnitude greater than the highest value reported in the literature to date (up to 15.6 microliter/mg) (Szoka, F.C. and Papahadjopoulos, D. (1978) Proc. Natl. Acad, Sci. U.S.A. 75, 4194-4198).

Chloroform↗
PubMed1981

On the activity and fidelity of chromatin-associated hepatic DNA polymerase-beta in aging murine species of different life spans.

Activity and accuracy of chromatin-directed DNA replication have been compared in young and aged Mus musculus and Peromyscus leucopus, two murine species with contrasting maximum lifespans. Chromatin isolated from livers of mature adults of both species copied efficiently exogenous DNA templates using predominantly DNA polymerase-beta. The DNA synthetic activity of liver chromatin remained constant in both species throughout their lifetimes. The fidelity of chromatin-directed poly [d(A-T)] synthesis was similar for the comparatively short-lived M. musculus and the relatively long-lived P. leucopus and remained unaltered in old animals. The fidelity of poly [d(A-T)] copying catalyzed by DNA polymerase-beta-dissociated from liver chromatin was comparable to that of the chromatin-directed synthesis. The dissociation enzymes did not exhibit diminished fidelity of poly [d(A-T)] synthesis with age. In all ages of both species examined, the murine liver DNA polymerase-beta, both chromatin-associated and solubilized, exhibited high error frequencies; approximately one dGMP was incorporated for every 500-1,000 complementary nucleotides polymerized. The relationship of these results to the accuracy of DNA replication and repair as a determinant of aging is considered.

Aging↗
PubMed1981

Effects of radical-scavenging enzymes and reduced oxygen exposure on growth and chromosome abnormalities of Werner syndrome cultured skin fibroblasts.

Two sister strains of skin fibroblast-like (FL) cells from a patient with Werner's syndrome (adult progeria) were grown in regular tissue culture medium or medium supplemented with the radical-scavenging enzymes superoxide dismutase and catalase. Differences in proliferative potential were shown to be due to strain variation rather than to treatment with the enzymes. Two independent strains were also grown in ambient (18%) or reduced (1%) concentrations of oxygen. All cultures (untreated, treated with enzymes, treated with reduced oxygen exposure) displayed the limited in vitro growth potential and cytogenetic abnormality characteristic of Werner's syndrome FL cells; thus the underlying defect in Werner's syndrome does not appear to be related to defective protection against or repair of damage by free radicals.

Catalase↗
PubMed1981

Systematic growth studies, cocultivation, and cell hybridization studies of Werner syndrome cultured skin fibroblasts.

The growth of 20 independently derived skin fibroblastlike (FL) cell strains from three individuals with Werner syndrome (adult progeria) was compared with the growth of ten FL cell strains from normal individuals. Population growth rates and total replicative life spans of Werner syndrome strains averaged 53% and 27%, respectively, of the growth rates and life spans of non-Werner strains. In the first few passages, four Werner syndrome strains demonstrated population growth rates in the low normal range, but the longest-lived Werner syndrome strain had only 75% of the total replicative potential of the shortest-lived normal strain. Exponential growth rates, cloning efficiencies, and saturation densities of Werner strains were also reduced, whereas cell attachment was normal. Viable cells (identified by dye exclusion) were maintained in post-replicative Werner syndrome and control cultures for periods of at least 10 months; there was no evidence of accelerated post-replicative senescence of cell death of Werner syndrome FL cells. Cocultivation of Werner syndrome and normal strains did not influence population growth rates of either strain. Two proliferating hybrid clones were obtained from fusions of normal and Werner syndrome FL cell strains and these hybrids displayed the reduced growth potential typical of Werner syndrome FL cells. These studies confirm that low growth rate and sharply reduced replicative life span are characteristic of cultured skin FL cells from patients with Werner syndrome, and they suggest that these characteristics are not affected by complementation with non-Werner FL cells.

Cell Division↗
PubMed1981

Growth inhibition and morphologic modulation of human fibroblastlike cells by erythromycin.

In vitro exposures of mass cultures and clones of human diploid fibroblastlike cells to erythromycin, in concentrations of 50 to 400 micrograms/ml, results in increasing degrees of growth inhibition and augmented cell volume, with a shift toward larger proportions of cells of the epithelioid type and fewer of the fibroblast type. These alterations were reversed upon subculture in the absence of the antibiotic.

Cell Division↗
PubMed1981

Cytogenetics of Werner's syndrome cultured skin fibroblasts: variegated translocation mosaicism.

Skin fibroblast-like (FL) cells from patients with Werner's syndrome (adult progeria) regularly demonstrate frequent pseudodiploidy involving variable structural rearrangements that are clonal: variegated translocation mosaicism (VTM). Ninety-two percent of 1,538 metaphases from 29 independent strains derived from five patients with Werner's syndrome demonstrated this cytogenetic abnormality. In contrast, only eight (8.4%) of 95 non-Werner's syndrome FL cell cultures demonstrated VTM: seven with low-grade VTM (approximately 5% of 300 metaphases), and one with VTM affecting 90-100% of metaphases. Unlike the cytogenetic abnormalities observed in the terminal stages of normal FL cell cultures, VTM occurs throughout the entire lifespan of Werner's syndrome cultures. Ten of the identifiable break points in 1,005 banded metaphases accounted for 27% of all definable rearrangements. Baseline sister chromatid exchanges were not increased. Cocultivation of Werner's syndrome and normal strains did not induce VTM in the normal strain. The relationship between VTM and the reduced growth potential of Werner's syndrome FL cells is not yet understood, nor is the relationship between these in vitro abnormalities and the presumptive single gene defect that causes the progeroid clinical manifestations of Werner's syndrome.

Adolescent↗
PubMed1981

Evidence of clonal attenuation, clonal succession, and clonal expansion in mass cultures of aging Werner's syndrome skin fibroblasts.

Skin fibroblast-like (FL) cells from patients with Werner's syndrome (adult progeria) demonstrate multiple, stable, structural chromosome rearrangements (variegated translocation mosaicism) which can be used to identify cytogenetically marked clones of cells within a mass culture. We have cytogenetically followed eight FL cell strains (from two patients) throughout their entire in vitro replicative lifespans, and we show a correlation between the expansion and attenuation of individual clones and the growth of the mass cultures. One strain, which was aged several times, demonstrated a generally reproducible pattern of clonal succession but, surprisingly, also demonstrated, in two parallel derivative cultures, the late emergence of two relatively rapidly growing clones that had not been observed in the parental culture. These observations suggest that clonal succession and clonal attenuation occur in mass cultures, as had been predicted on the basis of dilute-plating cloning experiments. Our results may have implications for models of in vitro cellular senescence. In addition, there are interesting parallels with the tissue hyperplasia associated with in vivo aging, and this observation is compatible with the suggestion that skin FL cells in vitro provide a model for hyperplasia in vivo.

Cell Cycle↗
PubMed1980

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta.

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.

Animals↗