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Biomedical subjects

R Holliday

Publications and source records attributed to R Holliday.

At least 109 records · Page 6Linked to original sources
PubMed1985

Differences in methylation on the active and inactive human X chromosomes.

Methylation of CCGG sites was examined in four regions of the X chromosome with four X-chromosome clones, three obtained by cloning random segments and one encoding a structural gene. In DNA from human peripheral blood cells unmethylated sites correlating with the inactive X chromosome were detected in the vicinity of two of the random clones and also in the vicinity of a cloned sequence of the X-linked phosphoglycerate kinase gene (PGK). The third random clone covered a region whose methylation pattern was unchanged between the active and inactive X chromosomes. Differential methylation at the sites detected appears to have no functional role in the maintenance of the inactive X chromosome since both active and inactive X chromosomes were found to be undermethylated in DNA from human lymphoblastoid cells.

Base Sequence↗
PubMed1985

Peripheral lymph flow in sheep with bacterial peritonitis: evidence for increased peripheral microvascular permeability accompanying systemic sepsis.

We studied the effects of systemic sepsis on peripheral microcirculatory fluid exchange by examining changes in flow (Qlymph) and lymph-to-plasma [L/P] total protein and albumin ratios from lymph draining, the efferent duct of a prefemoral lymph node in sheep, before and during surgically-induced peritonitis. After baseline study, peritonitis was produced by cecal ligation, perforation, and devascularization. By 24 hours blood cultures revealed a polymicrobial bacteremia. The hemodynamic response to the septic insult during the 72-hour study period was characterized by an increase in heart rate and an initial fall in stroke volume index; yet, the mean blood pressure remained unchanged from baseline levels throughout the study protocol. The intrapulmonary shunt fraction increased (p less than 0.05) by 48 hours, as did both the Qlymph (2.6 +/- 1.9 ml/hr to 6.8 +/- 4.6 ml/hr; p less than 0.05) and the calculated lymph albumin clearance (1.6 +/- 1.2 ml/hr to 3.1 +/- 1.7 ml/hr; p less than 0.05). Although the calculated serum to interstitial colloid osmotic pressure gradient fell (F = 4.37; p less than 0.04), both the [L/P] total protein and albumin ratios were unchanged from baseline throughout 72 hours of study. Further, [L/P] total protein ratios were unrelated to Qlymph (r = -0.20); as Qlymph (experimental/baseline) increased with sepsis, [L/P] total protein ratio (experimental/baseline) did not fall (r = +0.62). We therefore conclude that systemic sepsis, as represented by this model of bacterial peritonitis, results in increased peripheral microcirculatory fluid flux that is primarily a consequence of an increase in permeability of the peripheral microvascular exchanging membrane.

Animals↗
PubMed1985

Mechanisms for changing gene expression and their possible relationship to carcinogenesis.

Several genetic or epigenetic events can alter gene expression and we assess their importance in multistage carcinogenesis. Mutation and chromosome rearrangement can produce changes in DNA sequence which have been identified in some cancer cells. Recombination and non-disjunction can lead to the expression of recessive alleles. Overexpression of genes by amplification has also been observed in certain tumours. Heteroploidy, resulting from karyotypic instability, is a common feature of tumorigenicity. Although the pattern of DNA methylation is heritable, there is considerable evidence that genes can be activated by loss of methylation or inactivated by de novo methylation. Hypomethylation is frequently observed in tumour cells. Mutation and chromosome translocation can produce rare specific changes which may be important as initiating events. In contrast, the extent of karyotypic instability and hypomethylation shows some correlation with the stage of tumour progression, and these changes may be important in generating the phenotypic diversity which allows the selection of variants during neoplasia and metastasis. The striking differences between the frequencies of cellular transformation in rodents and man may be more easily explained by differences in the epigenetic controls of gene activity, than by abnormalities in DNA sequence.

Animals↗
PubMed1984

The biological significance of meiosis.

Four possible biological functions of meiosis are considered. First, the conventional view that it generates by recombination and sexual reproduction the genetic diversity on which natural selection can act. Second, that recombination at meiosis plays an important role in the repair of genetic defects in germ line cells. Third, that it is essential, at least in animals, for the reprogramming of gametes which give rise to the fertilized egg. Fourth, that it helps maintain the immortality of the germ line, possible by a process of rejuvenation involving the removal of faulty RNA and protein molecules, or by the elimination of defective meiocytes. A unified hypothesis is proposed which attempts to link these diverse functions. Evidence is now available which strongly indicates that the control of gene activity in higher organisms depends in part on the pattern of cytosine methylation in DNA, and that this pattern is inherited through the activity of a maintenance methylase. Epigenetic defects may arise by the loss of methyl groups which the methylase is unable to replace in somatic and also germ line cells, if de novo methylation cannot occur. There is also evidence that recombination at meiosis is largely confined to structural genes or adjacent DNA. It is proposed that the absence of a functionally important methyl group in a promotor or operater region produces a recombinator or signal for the initiation of recombination. The formation of hybrid DNA in this region then allows the lost methyl groups to be replaced by maintenance methylase activity. The removal of epigenetic defects by recombination during meiosis therefore becomes an essential part of a reprogramming and rejuvenation process. Assuming some epigenetic defects are nevertheless transmitted to the next generation, sexual reproduction and outbreeding would be advantageous because they provide the opportunity for their removal at the next meiosis. Inbreeding would be disadvantageous, because it increases the probability that epigenetic defects would become homozygous and could no longer be removed by recombination.

5-Methylcytosine↗
PubMed1983

Genetic effects on the longevity of cultured human fibroblasts. I. Werner's syndrome.

Fibroblast cultures were established from skin biopsies from 4 male Werner's syndrome patients, aged 45-50. Several subcultures were obtained from each primary outgrowth and these were passaged to the end of their in vitro lifespan. Their average longevity was significantly less than control skin fibroblasts, but there was extreme variability amongst parallel cultures. In the most thoroughly studied case, the longevity of 19 subcultures derived from a single biopsy varied from 5 to 26 passages, showing that there was considerable heterogeneity in the growth potential of the cells in the primary culture. Moreover, the growth rate from any one culture was not uniform, since long periods of slow growth were sometimes succeeded by much more rapid proliferation. These features of Werner's syndrome fibroblast populations are not seen in cultures from normal individuals. The longevity of fibroblasts from one progeria patient was also shown to be much shorter than controls. Metaphases from 3 Werner's patients demonstrated a much higher frequency of chromosome abnormalities than in normal fibroblasts and also provided evidence of subclones containing a characteristic 'marker' chromosome.

Adolescent↗
PubMed1983

Genetic effects on the longevity of cultured human fibroblasts. II. DNA repair deficient syndromes.

The lifespan of fibroblasts from genetic syndromes with reduced DNA repair or chromosome stability has been measured. Cells from Bloom's syndrome, Cockayne's syndrome, Fanconi's anaemia and 2 out of 3 cases of ataxia telangiectasia had a significantly reduced growth potential in comparison to controls. In each case the longevity of several parallel populations was measured and the greatest variability in lifespan was observed with Cockayne's syndrome cells. The fibroblasts from 1 ataxia telangiectasia patient and a Friedreich's ataxia patient grew to the passage levels seen in control cultures. The results suggest that repair processes are necessary for cells to achieve their maximum in vitro lifespan, and support the error theory rather than the programme theory of ageing.

Aged↗
PubMed1983

Genetic effects on the longevity of cultured human fibroblasts. III. Correlations with altered glucose-6-phosphate dehydrogenase.

The level of heat-labile glucose-6-phosphate dehydrogenase (G6PD) has been measured in skin fibroblast cultures from premature ageing or DNA repair deficient genetic syndromes. The short in vitro longevity of Werner's syndrome, progeria, Cockayne's syndrome, ataxia telangiectasia, Fanconi's anaemia, and Bloom's syndrome cultures was correlated with the appearance of a significant fraction of heat-labile enzyme. Long-lived control cultures contain a low level of altered enzyme until they become senescent. The evidence that heat-labile G6PD molecules are derived from errors in synthesis, or from other causes, is critically assessed. It is shown that normal cells grown in medium containing the antibiotic, paromomycin, which is known to reduce the fidelity of ribosomal translation, produce a significant fraction of altered G6PD.

Aged↗
PubMed1983

Genetic effects on the longevity of cultured human fibroblasts. IV. Enhanced growth potential of cystic fibrosis cells.

The in vitro longevity of skin fibroblasts in several parallel cultures from each of 4 cystic fibrosis (CF) patients and 3 control patients was measured by serial passages. The control cultures reached 51-56 passages before growth ceased. 1 CF strain achieved an average of 69 passages and another 64 passages. Both of these longevities are significantly greater than the controls. 2 CF strains had significantly shorter longevities than the controls. The results indicate that the CF gene is expressed in cultured fibroblasts and provide further evidence that there may be more than one form of the disease.

Adolescent↗
PubMed1983

Limited and unlimited growth of SV40-transformed cells from human diploid MRC-5 fibroblasts.

Human foetal lung strain, MRC-5, was treated with simian virus 40 and cultures were obtained that had many of the properties of transformed populations. In 10 experiments, only two produced permanent lines, designated MRC-5V1 and MRC-5V2, which have grown to passage 750 and 650, respectively. In all cases, the SV40-treated cultures acquired many of the features of transformation, including production of T-antigen, loss of contact-inhibition, and ability to grow in low concentrations of serum. The presence or absence of other transformed characteristics, such as altered morphology, abnormal karyotype or ability to grow in soft agar, can be used to distinguish between individual newly infected cultures. However, the cells invariably entered a period of slow growth, or crisis, and in eight experiments the cultures subsequently died without the emergence of a permanent line. The report that late-passage diploid cultures ae more easily transformed to permanent lines than young cultures has not been confirmed. MRC-5V1 initially had a sub-diploid chromosome number, but during serial passaging this gradually increased. MRC-5V2, which has a more extreme transformed phenotype than MRC-5V1, had a hyper-diploid chromosome number, which also increased during long-term growth. MRC-5V1 became polymorphic for glucose-6-phosphate dehydrogenase, as judged by the heat-lability and electrophoretic mobility of the enzyme. Fusions between MRC-5V1 and Lesch-Nyhan fibroblasts yielded hybrids with a limited lifespan, and certain sub-lines of MRC-5V1 also slowed down, exhibited characteristic signs of senescence and ceased to grow.

Bromodeoxyuridine↗
PubMed1982

Intracellular accumulation of a fluorescent derivative of paromomycin in human fibroblasts.

Human fetal lung fibroblasts grown in the presence of dansyl-paromomycin (DNS-Pm), a fluorescent derivative of the aminoglycoside antibiotic, paromomycin, probably accumulate DNS-Pm in the lysosomes. The intracellular concentration of DNS-Pm is proportional to the extracellular concentration and to the length of time cells are exposed to the compound. The accumulation of DNS-Pm by human fibroblasts continued to increase for several days, reaching a saturation after 7 days. The kinetic data are consistent with the establishment of a steady state in the cell between fluid-phase pinocytosis and exocytosis of DNS-Pm. About 80% of the intracellular DNS-Pm ws released in 24 hr when fresh medium without the analogue was added. The residual 20% remained within the cells, suggesting that it may be irreversibly bound to the lysosomes, endoplasmic reticulum, or ribosomes. The uptake of paromomycin by cells in culture may be a useful means to study error propagation during growth and lifespan of cells in vitro.

Chemical Phenomena↗