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Biomedical subjects

C B Harley

Publications and source records attributed to C B Harley.

At least 73 records · Page 4Linked to original sources
PubMed1987

Analysis of E. coli promoter sequences.

We have compiled and analyzed 263 promoters with known transcriptional start points for E. coli genes. Promoter elements (-35 hexamer, -10 hexamer, and spacing between these regions) were aligned by a program which selects the arrangement consistent with the start point and statistically most homologous to a reference list of promoters. The initial reference list was that of Hawley and McClure (Nucl. Acids Res. 11, 2237-2255, 1983). Alignment of the complete list was used for reference until successive analyses did not alter the structure of the list. In the final compilation, all bases in the -35 (TTGACA) and -10 (TATAAT) hexamers were highly conserved, 92% of promoters had inter-region spacing of 17 +/- 1 bp, and 75% of the uniquely defined start points initiated 7 +/- 1 bases downstream of the -10 region. The consensus sequence of promoters with inter-region spacing of 16, 17 or 18 bp did not differ. This compilation and analysis should be useful for studies of promoter structure and function and for programs which identify potential promoter sequences.

Base Sequence↗
PubMed1987

Loss of brown adipose tissue uncoupling protein mRNA on deacclimation of cold-exposed rats.

The effect of environmental temperature on the level of uncoupling protein mRNA from rat brown adipose tissue was examined using a cDNA probe. A 4.4 fold increase in the mRNA level was observed after 1 day exposure of rats to 6 degrees C, which was followed by a slow loss with longer times of exposure. When rats were returned to a thermoneutral environment, there was a dramatic loss of uncoupling protein mRNA within 1 day. Comparison wih poly(A)+ RNA levels suggest that the response to temperature is specific for uncoupling protein mRNA.

Acclimatization↗
PubMed1987

Expression of mRNA homologous to interleukin 1 in human epidermal cells.

Epidermal cells produce an interleukin 1 (IL-1)-related molecule known as epidermal cell-derived thymocyte activating factor (ETAF). Given their similar physical and biologic properties, it was presumed that ETAF would share homology to known IL-1 sequences. A murine IL-1 alpha complimentary DNA (cDNA) probe hybridized with a 2.1 kb RNA from the murine keratinocyte cell line PAMM 212. Further, a 24-base synthetic IL-1 beta probe complementary to a region 700 bases from the putative 5'-end of human IL-1 beta hybridized with 2.7- and 1.6-kb RNAs in human keratinocytes, monocytes, and promyelocytic leukemia cells. Reverse transcripts primed with the IL-1 beta oligonucleotide revealed major first-strand cDNAs from human keratinocyte COLO-16 cell mRNA and human monocyte mRNA of about 700 bases, consistent with the expected cDNA from IL-1 beta. A partial library constructed from this cDNA contained clones that hybridized with the IL-1 beta oligonucleotide. The 750 bp-IL-1 beta-like cDNA clones were not identical to IL-1 beta as judged by restriction mapping and Northern analysis. These cDNA clones hybridized with the 1.6-kb keratinocyte mRNA but not to the 1.6-kb monocyte nor to the 2.7-kb mRNA. The 1.6-kb keratinocyte mRNA may encode a new IL-1-like species.

Cloning, Molecular↗
PubMed1986

Ca2+ and phorbol ester synergistically induce HL-60 differentiation.

Exposure of HL-60 cells to subthreshold concentrations of TPA caused monocytic differentiation only when cells were cotreated with the Ca2+ ionophore A23187. Phorbol ester dose-response curves for growth arrest and enzymatic markers of differentiation were shifted to lower concentrations when the ionophore was present. Expression of a monocyte/granulocyte cell surface antigen also occurred only when cells were treated with both agents. Similar effects were seen with other active but not inactive phorbol esters and with another Ca2+ ionophore. The Ca2+ component of phosphoinositide-based signalling may thus play a role in HL-60 differentiation.

Antigens, Surface↗
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↗
PubMed1983

Some aspects of cellular aging.

Studies were carried out on cultured human fibroblasts in order to elucidate the biology of aging and the origins of age-dependent diseases. The replicative life span of cultures was inversely proportional to the chronological age of the tissue donor, and cultures derived from subjects with two inherited disorders of premature aging, progeria and Werner syndrome, had more severely impaired growth capacity. Studies on circular outgrowths whereby cell division is restricted to a circumferential rim of cells indicated that the replicative life span is controlled by a mitotic counter to a critical limit. The response of progeria cells to a hormone preparation with insulin-like activity was decreased, while in normal cells this decrease occurred as a function of passage level with a "shift to the right" of the dose-response curve. Cyclic AMP content of fibroblasts at late passage fell in response to PGE1 stimulation but rose in response to epinephrine, likely due to altered expression of genes for the receptors for each of these two hormones. This system of cultured human fibroblasts is useful in explaining various concomitants of biological aging including decreased tissue cellularity and impaired hormone and drug responsiveness.

Aging↗
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↗
PubMed1981

Decreased sensitivity of old and progeric human fibroblasts to a preparation of factors with insulinlike activity.

To determine whether old cells have a reduced response to a preparation of factors from human plasma with insulinlike activity (ILA), we analyzed the response to ILA of early and late passage human fibroblasts from young, old, and progeric donors in the acute stimulation of [3H]2-deoxy-D-glucose (2dG) uptake and the delayed stimulation of [3H]thymidine (TdR) incorporation into DNA. The ILA concentration required to produce equivalent, relative stimulation of TdR incorporation was increased two- to three-fold in late passage cells and cells from old and progeric donors (P less than 0.01). 50 and 95% of maximal stimulation (ILA50, ILA95) was achieved by 0.26 +/- 0.07 and 1.38 +/- 0.13 ng insulin equivalents/ml (mean +/- SD) respectively, in cells from young adults at early passage. Corresponding values were 0.54 +/- 0.05 and 2.90 +/- 0.25 in cells from old donors; greater than 0.9 +/- 0.1 and greater than 3.1 +/- 0.1 in cells from a 9-yr-old progeric donor; and 0.4 +/- 0.05 and 1.1 +/- 0.04 in cells from normal children (9-13 yr). For two cell strains from young adults, ILA50 and ILA95 were 0.30 +/- 0.02 and 1.0 +/- 0.3 ng eq/ml at 30% of their in vitro lifespan completed (%LC) and these values increased at rates of 0.005 ng eq/ml per %LC and 0.04 ng eq/ml per %LC, respectively. The mean stimulation of 2dG uptake ratio (ILA/control) decreased from early to late passage from 2.1 +/- 0.6 to 1.3 +/- 0.1 in young adult donors (P less than 0.05), but there were no significant differences between young and old donors at either early or late passage. The mean stimulation ratio in progeric cells (1.2 +/- 0.2) did not change with in vitro passage, but was significantly lower than that of age-matched normal cells (2.1 +/- 0.8, P less than 0.001). In progeria cells, the reduced stimulation of 2dG uptake upon addition of ILA was due to an increased basal rate of uptake (0.19 +/- 0.01 pmol [3H]2dG/min per mg protein vs. 0.13 +/- 0.01 in age-matched normal cells), and not to a decline in the maximal rate of uptake (0.26 +/- 0.01 vs. 0.27 +/- 0.02, respectively). Similar results were found for in vitro aging in cells from an old donor.

Adolescent↗
PubMed1980

Retesting the commitment theory of cellular aging.

The commitment theory of human fibroblast aging predicts that 55 percent of cells will be nondividing at the middle to late stages of the replicative lifespan; in the present study, however, fewer than 10 percent were nondividing. The fact that no immortal diploid cells have yet been reported is also at odds with the theory. Available data on the variable life-span of clones and mass cultures, the dependence of longevity on population size, and the predominance of certain cell types at termination of a culture are compatible with simpler theories, which support the idea that the limited replicative life-span of diploid fibroblasts is a valid model for organismic aging.

Cell Differentiation↗
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↗
PubMed1979

In vitro studies of age-associated diseases.

We have carried out studies on cultured human fibroblasts in an attempt to trace the origins of age-dependent disorders to the cellular and molecular levels. Three interrelated areas are discussed. First, skin donors with diabetes mellitus (a disease complex that features inappropriate hyperglycemia) produce cultured fibroblasts with a moderate reduction in growth capacity, while two inherited disorders of inappropriate hyperglycemia and premature aging, progeria and Werner syndrome, yield fibroblast cultures with more severely impaired growth capacity. Second, there is a decreased response of progeria level and donor age; evidence is presented that this defective hormone responsiveness in aging cells may reside at the hormone receptor on the surface membrane, the cyclic AMP system, the intracellular enzymatic machinery, or all of these sites. Third, tissue factor, a procoagulant that activates the extrinsic clotting mechanism, is more abundant in cells from the premature aging syndromes of progeria and Werner syndrome. Fibroblast aging in vitro may help to explain various concomitants of normal aging and diabetes mellitus including cell dropout, impairment of hormone responsiveness, and increased atherothrombosis.

Aging↗
PubMed1978

Cultured human fibroblasts: distribution of cell generations and a critical limit.

In circular outgrowths of human skin fibroblasts we found that mitotic cells at the circumference had consumed more of their replicative lifespan than cells located more centrally. The lifespan remaining in cells at a given radial position could be predicted by determining their generation level based on the rate at which the outgrowth expanded and the cell doubling time. The data also show that outgrowths contain a heterogeneous mixture of cells described by a linear distribution of generations which can account for the variable replicative capacity observed in clones and the exponential increase in the fraction of nondividing cells with serial passage. These results support the concept that a critical limit of cell divisions determines the replicative lifespan.

Cell Division↗