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Biomedical subjects

J W Pollard

Publications and source records attributed to J W Pollard.

At least 109 records · Page 6Linked to original sources
PubMed1987

Estrogens and cell death in murine uterine luminal epithelium.

The luminal epithelium of adult ovariectomized mice responds to estradiol-17 beta with a synchronised wave of DNA synthesis and mitosis. Estriol, however, although producing a similar DNA-synthetic and mitotic response fails to cause an increase in cell number owing to a wave of cell death occurring at mitosis. In the present study it was shown that cells died by two different routes. The majority died by apoptosis but, unusually, a minority also died by necrosis. In the apoptotic cells the cytoplasm became dense, the endoplasmic reticulum and nuclear cisternae dilated; chromatin became marginated the nucleus shrank and became deeply infolded and contorted. Apoptosis, however, was uncharacteristic in that the nucleus failed to fragment, form caps or show disruption before the cells died by membrane rupture. Furthermore, the cells were frequently lost in sheets from the epithelium into the lumen. Part of the biochemical explanation for this onset of cell death comes from the accelerated loss from the tissue of estriol when compared to estradiol-17 beta. This resulted in a decline in protein and rRNA biosynthesis and a failure to complete ribosomal maturation. Evidence in favour of this explanation came from experiments that showed a return to the estradiol-17 beta level of response and an inhibition of cell death when the occupancy of the estriol receptor was maintained.

Animals↗
PubMed1987

Hypothesis: somatic hypermutation by gene conversion via the error prone DNA----RNA----DNA information loop.

A mechanism to explain somatic hypermutation in immunoglobulin variable region genes is proposed employing polynucleotide information transfer through the error prone DNA----RNA----DNA loop. During transcription of the rearranged V-region, the primary transcript undergoes either inappropriate termination, cleavage, or reverse transcriptase priming allowing a V-region specific reverse-transcriptase-integrase complex to synthesize a DNA copy of the rearranged V-region and integrate it, by homologous recombination, back into the normal chromosomal site. Some consequences and predictions of the hypothesis are discussed.

Animals↗
PubMed1986

Regulation of colony-stimulating factor 1 during pregnancy.

Pregnancy results in an elevation in serum and tissue concentrations of the mononuclear phagocytic growth factor, CSF-1 (colony-stimulating factor 1). These increases are associated with an increase in the number of monocytes in the circulation, and with increases in the number of splenic macrophage precursors. In contrast to the approximately 2-fold elevation of the CSF-1 concentrations in serum and most tissues, pregnancy results in a 1,000-fold increase in the concentration of uterine CSF-1. The roughly fivefold elevation in uterine CSF-1 concentration observed at day 5 of pregnancy could be mimicked by administration of chorionic gonadotrophin in intact but not ovariectomized mice. These dramatic changes in uterine CSF-1 concentrations may indicate a role for CSF-1 in the regulation of nonmononuclear phagocytic cell types.

Animals↗
PubMed1986

Regulation of polypeptide chain initiation and activity of initiation factor eIF-2 in Chinese-hamster-ovary cell mutants containing temperature-sensitive aminoacyl-tRNA synthetases.

The regulation of polypeptide chain initiation has been investigated in extracts from a number of well-characterized Chinese hamster ovary (CHO) cell mutants containing different temperature-sensitive aminoacyl-tRNA synthetases. These cells exhibit a large decline in the rate of initiation when cultures are shifted from the permissive temperature of 34 degrees C to the non-permissive temperature of 39.5 degrees C. During a brief incubation with [35S]Met-tRNAMetf or [35S]methionine, formation of initiation complexes on native 40S ribosomal subunits and 80S ribosomes is severely impaired in extracts from the mutant cell lines exposed to 39.5 degrees C. Wild-type cells exposed to 39.5 degrees C do not show any inhibition of protein synthesis or initiation complex formation. Inhibition of formation of 40S initiation complexes in the extracts from mutant cells, incubated at the non-permissive temperature, is shown to be independent of possible changes in mRNA binding or the rate of polypeptide chain elongation and is not due to any decrease in the total amount of initiation factor eIF-2 present. However, assays of eIF-2 X GTP X Met-tRNAMetf ternary complex formation in postribosomal supernatants from the temperature-sensitive mutants reveal a marked defect in the activity of eIF-2 after exposure of the cells to 39.5 degrees C and addition of exogenous eIF-2 to cell-free protein-synthesizing systems from cells incubated at 34 degrees C and 39.5 degrees C eliminates the difference in activity between them. The activity of the initiation factor itself is not directly temperature-sensitive in the mutant CHO cells. The results suggest that the activity of aminoacyl-tRNA synthetases can affect the ability of eIF-2 to bind Met-tRNAMetf and form 40S initiation complexes in intact cells, indicating a regulatory link between polypeptide chain elongation and chain initiation.

Amino Acyl-tRNA Synthetases↗
PubMed1986

c-rasH and ornithine decarboxylase are induced by oestradiol-17 beta in the mouse uterine luminal epithelium independently of the proliferative status of the cell.

Oestradiol-17 beta (E2) treatment of the ovariectomized mouse results in a synchronised wave of cell proliferation in the uterine luminal epithelium. At the peak of DNA synthesis the mRNA level of the c-rasH proto-oncogene and ornithine decarboxylase were significantly increased. Progesterone treatment completely inhibits the E2 induced wave of DNA synthesis but does not greatly influence the level of these 2 mRNAs. Thus in the uterine luminal epithelium E2 regulates the level of ornithine decarboxylase and c-rasH independently of cell proliferation.

Animals↗
PubMed1986

High-frequency transfection of CHO cells using polybrene.

High-frequency transfection of CHO cells has been achieved for several plasmids, a cosmid library, and genomic DNA using Polybrene and dimethyl sulfoxide. All plasmid transfectants examined were stable and exhibited plasmid sequences in genomic DNA. The method is simple, reproducible, and succeeded with several independent CHO clones in the presence or the absence of carrier DNA, even at very low concentrations of plasmid DNA.

Animals↗
PubMed1985

Protein synthetic fidelity in aging human fibroblasts.

The fidelity of protein synthesis was measured in human diploid skin fibroblasts as a function of passage level ("aging in vitro") and physiological age of tissue donor ("aging in vivo") using two different test systems. First, in cell-free extracts the ratio of delta leu/delta phe incorporation into peptide linkage following in the latter case using cells derived from elderly normal donors and from subjects with the premature aging disorders of Hutchinson-Gilford progeria and the Werner syndrome. Similar results were obtained using a second system of intact cells whereby histidine starvation induces quantifiable satellite spots resolved by two dimensional electrophoresis on polyacrylamide gels on the acidic side of the native actin species due to substitution of the neutral amino acid glutamine for the basic histidine. In fact, error frequencies appeared to decrease during aging in vitro, likely due to selection for clonal subpopulations with the highest fidelity of protein synthesis. The only increases were seen in the intact cell system where SV40-transformed cells showed a three-to-five fold greater error frequency compared to nontransformed fibroblasts. In total, these data fail to support the error catastrophe theory of cellular aging.

Adult↗
PubMed1985

Cell growth and cell proliferation may be dissociated in the mouse uterine luminal epithelium treated with female sex steroids.

The mouse uterine epithelium under various hormonal regimes is a good system to identify biochemical events associated with cell growth, DNA synthesis and cell division. This is because estradiol-17 beta stimulates the cells to undergo a synchronized wave of DNA synthesis and cell division. Estriol, on the other hand, also stimulates DNA synthesis but because of the rapid loss of this hormone from the tissue some of the cells abort, giving a constant epithelial cell number. Three days of progesterone pretreatment, however, completely suppresses the estradiol-17 beta-induced wave of DNA synthesis and cell proliferation. Using these hormonal treatments we have shown that both estradiol-17 beta and estriol stimulate protein and rRNA synthesis with the concomitant increase of protein and rRNA per mg of DNA. These macromolecules accumulated in direct proportion to the fraction of cell committed to DNA synthesis. Estriol, however, did not sustain the growth responses and at the peak of DNA synthesis both rRNA and protein synthesis had returned to control levels. Progesterone pretreatment, despite inhibiting the proliferative response, failed to inhibit any of the estradiol-17 beta-induced increases in protein and rRNA synthesis. Indeed 12 h after estradiol-17 beta injection the cells had identical protein and rRNA contents, regardless of whether they had been exposed to progesterone or not. The present data therefore suggests that in the uterine epithelium cell growth as defined by protein and rRNA accumulation and DNA synthesis represents two independently regulated pathways.

Animals↗
PubMed1984

Selective synthesis of mitochondrial proteins by Chinese hamster ovary cells severely starved for various amino acids.

Chinese hamster ovary (CHO) cells were subjected to severe amino acid starvation for histidine, leucine, methionine, asparagine, tyrosine, glutamine, valine, and lysine, using amino acid analogs or mutations in specific aminoacyl-tRNA synthetases. At protein synthetic rates of less than 5%, in all cases, the newly synthesized proteins were found on two-dimensional electrophoretic gels to consist of a few intensely labeled spots, with the exception of lysine. This pattern could also be produced by strong inhibition of cytoplasmic protein synthesis with cycloheximide, and was abolished by preincubation with the mitochondrial protein synthesis inhibitor chloramphenicol. It appears therefore that the spots represent mitochondrial protein synthesis and that animal cells must have separate aminoacyl-tRNA synthetases for mitochondrial tRNAs corresponding to all these amino acids except, possibly, for lysine.

Amino Acids↗
PubMed1984

Life-threatening vagal reactions associated with percutaneous cholecystostomy.

In four of our initial five cases of percutaneous cholecystostomy, performed under ultrasound guidance for septicemia due to suspected cystic duct obstruction and hydrops of the gallbladder, patients suffered profound vagal reactions with severe hypotension and bradycardia. One patient had cardiac arrest. The patients had experienced recent myocardial infarction, ventricular ectopy, or septicemia, and each had a greatly distended gallbladder. The reactions occurred immediately, though in the first patient recognition was delayed. One patient had infected bile, three had obstructing cystic duct stones with inflammatory response, and one had a sterile and nonobstructed gallbladder. All patients survived the acute insult. Treatment consisted of minimizing intravisceral manipulations, administration of intravenous atropine, fluids, antibiotics, and dopamine. Atropine administered prior to the procedure appeared to blunt the reaction in one patient. Two patients died within a week of other causes. At autopsy neither peritoneal cavity had bile staining. The other patients had follow-up ultrasound examinations; no intraperitoneal fluid collections were seen.

Aged↗
PubMed1984

Do HMOs produce specific services more efficiently?

Previous research on the effects of HMOs on health care costs has concentrated on aggregate costs and resource use and has shown that HMOs result in lower costs. The only consistent sources of the cost savings are the lower hospital admission rates and hospital lengths of stay for HMO enrollees. This paper contains the results of an investigation of whether HMOs can more efficiently produce a given service. Four common inpatient procedures were analyzed to determine whether there were any differences in resource consumption and overall costs for HMO patients than for fee-for-service patients. Although significant levels of resource savings were found for various procedures for HMO enrollees, these savings did not always result in lower overall costs.

Cost Control↗
PubMed1982

Is transformation associated with an increased error frequency in mammalian cells?

Acute starvation of mammalian cells for amino acids results in translational errors that may be detected by two-dimensional polyacrylamide gel electrophoresis. Using this as an assay for error frequency in mammalian cells, we investigated the hypothesis that neoplastic transformation was associated with an increased error frequency which in turn leads to an increased mutation rate and a decreased efficiency of regulatory controls (phenomena of tumor progression). Although we found that transformation was not always associated with an increased level of mistranslation we showed that SV40 transformation increased the level of translational errors in all cell types tested.

Amino Acids↗
PubMed1980

Mammalian cells do not have a stringent response.

A key attribute of the stringent response of bacteria is the rapid inhibition of ribosomal RNA synthesis mediated by unusual nucleotides in respnse to uncharged tRNA. The question as to whether mammalian cells show a stringent response analogous to that of bacteria was critically tested by the effective rapid amino acid starvation of both normal and transformed cells. Rapid starvation giving a high proportion of uncharged tRNA for leucine was produced within 7 minutes of expression of a nonleaky ts leucyl tRNA synthetase mutation in transformed CHO cells (tsH1) and in its normal growth control revertant (L-73). To control for the effect of temperature alone, ts revertants of tsH1 and L-73 were included in the study, and to control for effects due simply to the inhibition of protein synthesis, the translational elongation inhibitor cycloheximide was used. In addition, rapid starvation for histidine was effected by incubation of both the CHO cell lines and of freshly explanted normal Chinese hamster embryo fibroblasts in histidine-free medium containing high concentrations of histidinol. The rate of preribosomal RNA synthesis and the extent of its maturation to mature rRNA was measured using (3H-methyl) methionine as a donor of methyl groups during synthesis and methylation of pre-rRNA. There was no effect on pre-rRNA synthesis of the rapid generation of uncharged tRNA for 45 minutes for any of the cell types tested. A nonspecific inhibition of maturation of 18S rRNA and late (3 hour) inhibition of pre-rRNA synthesis was observed, but could be mimicked by the inhibition of protein synthesis to comparable levels with cycloheximide. Less severe amino acid starvation resulting in a more physiological inhibition of protein synthesis to 30% also had no specific effect on pre-rRNA synthesis and maturation. Intracellular nucleotide pools were also examined for the appearance of unusual nucleotides such as guanosine tetraphosphate or pentaphosphate and for changes in the levels of normal nucleotides after severe amino acid starvation. No such changes could be detected. We conclude that although mammalian cells may have some biochemical reactions which respond to uncharged tRNA, they do not possess a macromolecular control system analogous to the stringent response of bacteria.

Amino Acids↗
PubMed1980

Protein synthetic errors do not increase during aging of cultured human fibroblasts.

To test the error catastrophe theory of aging we determined the error frequency of protein synthesis in several strains of cultured human fibroblasts at early and late passage. Error rates were calculated from analysis of native and substituted actins on two-dimensional gels of cellular proteins after induction of mistranslation by histidine starvation in the presence of histidinol. Early-passage cells from fetal, young, and old donors and cells from subjects with the Hutchinson-Gilford and Werner syndromes of accelerated aging had similar error frequencies. Late-passage cells from fetal, young, and old normal donors had similar or lower error frequencies than corresponding early-passage cells. No correlation was observed between error frequency, donor age, or maximal life span in vitro. We also examined an immortal cell line, simian virus 40-transformed W138 fibroblasts. These cells had a significantly elevated rate of mistranslation (2.8 +/- 0.2 x 10(-4))(+/- SEM) compared to their untransformed counterpart WI38 (0.6 +/- 0.1 X 10(-4)) or all diploid cells combined (1.1 +/- 0.1 x 10(-4)). Taken together, the data fail to support the error catastrophe theory of aging.

Actins↗