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C B Harley

Publications and source records attributed to C B Harley.

At least 19 recordsLinked to original sources
PubMed1992

Telomere length predicts replicative capacity of human fibroblasts.

When human fibroblasts from different donors are grown in vitro, only a small fraction of the variation in their finite replicative capacity is explained by the chronological age of the donor. Because we had previously shown that telomeres, the terminal guanine-rich sequences of chromosomes, shorten throughout the life-span of cultured cells, we wished to determine whether variation in initial telomere length would account for the unexplained variation in replicative capacity. Analysis of cells from 31 donors (aged 0-93 yr) indicated relatively weak correlations between proliferative ability and donor age (m = -0.2 doubling per yr; r = -0.42; P = 0.02) and between telomeric DNA and donor age (m = -15 base pairs per yr; r = -0.43; P = 0.02). However, there was a striking correlation, valid over the entire age range of the donors, between replicative capacity and initial telomere length (m = 10 doublings per kilobase pair; r = 0.76; P = 0.004), indicating that cell strains with shorter telomeres underwent significantly fewer doublings than those with longer telomeres. These observations suggest that telomere length is a biomarker of somatic cell aging in humans and are consistent with a causal role for telomere loss in this process. We also found that fibroblasts from Hutchinson-Gilford progeria donors had short telomeres, consistent with their reduced division potential in vitro. In contrast, telomeres from sperm DNA did not decrease with age of the donor, suggesting that a mechanism for maintaining telomere length, such as telomerase expression, may be active in germ-line tissue.

Base Sequence↗
PubMed1992

Telomere end-replication problem and cell aging.

Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.

Adult↗
PubMed1992

Release of a transforming growth factor (TGF)-beta 2-related suppressor factor from postimplantation murine decidual tissue can be correlated with the detection of a subpopulation of cells containing RNA for TGF-beta 2.

Postimplantation murine decidual tissue from allopregnant C3H mice has been shown to release in vitro a potent immunosuppressive factor closely related to transforming growth factor (TGF)-beta 2 but slightly lower in apparent molecular weight. Decidual suppressor factor (DSF) activity was first detected in decidual tissue supernatant at day 9.5 of gestation and reached a plateau by day 10.5 to 12.5. By Northern analysis of decidual and placental tissue with a simian TGF-beta 2 probe, two characteristic TGF-beta 2 mRNA transcripts were detected in decidual tissue. In situ hybridization analysis of C3H implant sites, with the simian (pGEM-G1G2) TGF-beta 2 riboprobe, revealed a small population of TGF-beta 2+ cells localized to postimplantation decidua basalis and metrial gland cell area after day 8.5. On and before day 8.5, when DSF was not detectable, few TGF-beta 2 mRNA+ cells were detected. To test for TGF-beta release in situ, sections of uterine tissue were stained with antibody specific for TGF-beta 2, that identified DSF in Western blots. In postimplantation tissues (day 9.5, 12.5) patchy anti-TGF-beta 2 staining was seen over decidual tissue. Before day 9.5, slight and diffuse staining over decidual tissue was present with more marked staining of extradecidual tissue. Very little staining was noted over day 9.5 decidual tissue by using anti-TGF-beta 1 antibody as a control; however, some staining was seen over postimplantation fetal trophoblast and myometrial tissue. Fractionation of disaggregated postimplantation decidua by velocity sedimentation revealed that TGF-beta 2 mRNA+ cells were predominantly small and sedimented in the same fraction(s) as those cells previously shown to release DSF in vitro. Thus, the release of TGF-beta 2 related DSF correlates with the in situ detection of TGF-beta 2 mRNA and the in situ release of TGF-beta 2 peptide. These studies suggest that DSF may be a form of TGF-beta 2 released by a population of small lymphocytic decidual suppressor cells.

Animals↗
PubMed1992

Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.

Loss of telomeric DNA during cell proliferation may play a role in ageing and cancer. Since telomeres permit complete replication of eukaryotic chromosomes and protect their ends from recombination, we have measured telomere length, telomerase activity and chromosome rearrangements in human cells before and after transformation with SV40 or Ad5. In all mortal populations, telomeres shortened by approximately 65 bp/generation during the lifespan of the cultures. When transformed cells reached crisis, the length of the telomeric TTAGGG repeats was only approximately 1.5 kbp and many dicentric chromosomes were observed. In immortal cells, telomere length and frequency of dicentric chromosomes stabilized after crisis. Telomerase activity was not detectable in control or extended lifespan populations but was present in immortal populations. These results suggest that chromosomes with short (TTAGGG)n tracts are recombinogenic, critically shortened telomeres may be incompatible with cell proliferation and stabilization of telomere length by telomerase may be required for immortalization.

Cell Transformation, Neoplastic↗
PubMed1992

Identification and characterization of a partial cDNA expressing interleukin-1-like activity in keratinocytes.

A partial cDNA has been isolated from the human keratinocyte cell line COLO-16 which is distinct from either IL-1 alpha or beta and encodes a protein which displays some of the biological properties associated with IL-1. The 720 bp partial cDNA hybridized on Northern blots of COLO-16 mRNA to a 1.6 kbp message of low abundance. Expression of the partial cDNA in COS-1 cells resulted in activity in three IL-1 assays: thymocyte co-stimulation, D10.G4.1 T-cell stimulation and fibroblast proliferation. Antisera generated against synthetic peptides based on inferred protein sequence from the cDNA reacted with a 20 kDa and 30 kDa species in both the COLO-16 cell line and PMA-stimulated normal human keratinocytes. These novel species were also present in PMA-stimulated and unstimulated human dermal fibroblasts and human T-cell lines.

Amino Acid Sequence↗
PubMed1992

Eosinophils in chronically inflamed human upper airway tissues express transforming growth factor beta 1 gene (TGF beta 1).

Transforming growth factor beta (TGF beta) is a multifunctional protein which has been suggested to play a central role in the pathogenesis of chronic inflammation and fibrosis. Nasal polyposis is a condition affecting the upper airways characterized by the presence of chronic inflammation and varying degrees of fibrosis. To examine the potential role of TGF beta in the pathogenesis of this condition, we investigated gene expression and cytokine production in nasal polyp tissues as well as in the normal nasal mucosa. By Northern blot analysis using a porcine TGF beta 1 cDNA probe, we detected TGF beta 1-specific mRNA in nasal polyp tissues, as well as in the tissue from a patient with allergic rhinitis, but not in the normal nasal mucosa. By the combination of tissue section staining with chromotrope 2R with in situ hybridization using the same TGF beta 1 probe, we found that approximately 50% of the eosinophils infiltrating the polyp tissue express the TGF beta 1 gene. In addition, immunohistochemical localization of TGF beta 1 was detected associated with extracellular matrix as well as in cells in the stroma. These results suggest that in nasal polyposis where eosinophils are the most prevalent inflammatory cell, TGF beta 1 synthesized by these cells may contribute to the structural abnormalities such as stromal fibrosis and basement membrane thickening which characterize this disease.

Chronic Disease↗
PubMed1991

Deproteination of nucleic acids by filtration through a hydrophobic membrane.

To remove or inactivate an enzyme from DNA in multistep procedures in molecular biology, it is often necessary to phenol extract the solution, followed by chloroform extraction and ethanol precipitation. In addition to being time-consuming and hazardous, there can be significant loss of DNA with this procedure, especially when small volumes or amounts of DNA are being used. We have found that filtering analytical reaction mixtures through a hydrophobic membrane specifically to remove protein is a rapid alternative to phenol extraction. Within broad limits commonly encountered in molecular biology, filtration through a polyvinylidene difluoride membrane quantitatively removes a variety of enzymes without significant loss of double-stranded nucleic acid.

Alkaline Phosphatase↗
PubMed1990

Fetal bovine serum contains an inhibitor of interleukin-1.

The keratinocyte cell line COLO-16 constitutively produces factors with interleukin-1 (IL-1) activity including IL-1 alpha and IL-1 beta. IL-1 activity assayed by thymocyte proliferation from cell supernatants was 20-50% less if cells were maintained in media containing 10% fetal bovine serum (FBS) compared to media without serum 24 h prior to harvest. The increased IL-1 activity in supernatants from cells in serum free media was not due to increased cellular levels of IL-1 alpha or IL-1 beta mRNA. Similarly, IL-1 activity recovered from conditioned supernatants of COS cells transfected with expression vectors containing IL-1 beta cDNA was approximately 22-45% less in cells grown in 20% FBS medium compared to similar cultures grown for 3 days post transfection in 1% FBS. When serial dilutions of recombinant IL-1 were made in buffer containing 10% FBS and assayed by a thymocyte proliferation method, a 30-50% decrease in activity was observed. IL-1 activity was also measured by its ability to induce prostaglandin E2 synthesis by fibroblasts. When COS conditioned supernatants were applied to fibroblast cultures there was 30% less prostaglandin E2 activity from fibroblasts treated with COS supernatants containing 20% FBS, compared to supernatants containing 1% FBS. The inhibitor molecule was partially purified by gel filtration and found to have a molecular weight of approximately 85,000. The presence of FBS in cell-conditioned media significantly reduces the sensitivity of IL-1 detection by bioassay techniques.

Animals↗
PubMed1990

Telomeres shorten during ageing of human fibroblasts.

The terminus of a DNA helix has been called its Achilles' heel. Thus to prevent possible incomplete replication and instability of the termini of linear DNA, eukaryotic chromosomes end in characteristic repetitive DNA sequences within specialized structures called telomeres. In immortal cells, loss of telomeric DNA due to degradation or incomplete replication is apparently balanced by telomere elongation, which may involve de novo synthesis of additional repeats by novel DNA polymerase called telomerase. Such a polymerase has been recently detected in HeLa cells. It has been proposed that the finite doubling capacity of normal mammalian cells is due to a loss of telomeric DNA and eventual deletion of essential sequences. In yeast, the est1 mutation causes gradual loss of telomeric DNA and eventual cell death mimicking senescence in higher eukaryotic cells. Here, we show that the amount and length of telomeric DNA in human fibroblasts does in fact decrease as a function of serial passage during ageing in vitro and possibly in vivo. It is not known whether this loss of DNA has a causal role in senescence.

Aging↗
PubMed1990

Reiterative copying by E.coli RNA polymerase during transcription initiation of mutant pBR322 tet promoters.

The major in vitro transcripts from the tet promoter of pBR322 derivatives pTA22 and pTA33 have heterogeneous 5' ends consisting of variable lengths of oligo(A). Their structure is 5'pppAnU..., where n ranges from 1 to greater than 12, but the template strand can encode at most four A residues at the site of transcription initiation. The abundance of additional A residues at the 5' end of the pTA22 and pTA33 tet transcripts could be reduced by elevating the concentration of UTP, but even at high concentrations (greater than 1 mM) non-cognate A residues were still observed. Aberrant initiation was not artifactual since the major and minor transcripts of the pBR322 tet promoter region, and other transcripts arising from minor promoters on pTA22 or pTA33 DNA all had unique 5' termini. Mixing experiments showed that RNA polymerase did not utilize pppA2-4-OH produced by abortive initiation as primers. The data suggest that the initial nascent RNA chain 'slips' in the 5' direction during elongation opposite T4 on the template strand causing RNA polymerase to reiteratively add A residues to the 5' end of the transcript. The generality and possible significance of this mechanism is discussed.

Base Sequence↗
PubMed1990

Expression of interleukin-1 beta in a human keratinocyte cell line.

The human keratinocyte cell line COLO-16 expresses mRNA homologous to human IL-1 alpha and IL-beta (transcript sizes 2.3 and 1.6 kb, respectively). A 1.2 kbp cDNA was selected with a human IL-1 beta probe from a lambda gt11 library constructed using poly A+ RNA from COLO-16 cells. Sequence analysis revealed that this cDNA was nearly identical to the 3' 1.2 kb of human monocyte IL-1 beta. When this cDNA was expressed in COS cells using a mammalian expression vector, IL-1 activity was detected in the cell conditioned supernatants using assays for D10-T-cell, thymocyte and fibroblast proliferation. Western analysis of lysates from COS cells transfected with this clone revealed the presence of a -17 kDa protein which reacted with antisera to human IL-1 beta. This protein was the same size as the processed form of IL-1 beta present in COLO-16 cells suggesting that this cDNA encodes the mature form of IL-1 beta. COLO-16 cells contain proteins of -30 kDa and 17 kDa which are immunoreactive with specific antisera for human IL-1 alpha and human IL-1 beta. Despite the presence of four-fold greater amounts of immunoreactive IL-1 beta protein than IL-1 alpha in cell lysates, all the IL-1 activity in the lysate could be neutralized by antisera to IL-1 alpha. IL-1 beta comprised only 25% of the IL-1 activity in the cell-conditioned media, all remaining activity was neutralized by antisera to IL-1 alpha. Whereas IL-1 alpha protein in both cell lysates and conditioned supernatants was predominantly in the processed -17 kDa form, IL-1 beta proteins were primarily of the processed and inactive 30kDa species. This apparent inability of keratinocytes to process IL-1 beta may explain our observations that the IL-1 activity secreted by COLO-16 cells is principally due to IL-1 alpha.

Amino Acid Sequence↗
PubMed1989

Metallothionein mRNA and protein induction by cadmium in peripheral-blood leucocytes.

To help to evaluate the role of metallothionein (MT) in peripheral-blood leucocytes, we examined MT protein and mRNA levels in these cells before and after exposure to CdCl2 in culture. Protein was assayed by 109Cd2+ binding, and RNA by dot-blot hybridization. MT was induced in both lymphocytes and adherent monocytes about 10-fold with a 12 h exposure to 10 microM-CdCl2, but absolute levels were 3-fold higher in monocytes: 57 x 10(5) (+Cd2+) versus 6 x 10(5) (-Cd2+) molecules/cell for monocytes; 18 x 10(5) (+Cd2+) versus 2 x 10(5) (-Cd2+) for lymphocytes. Polymorphonuclear cells expressed relatively little MT (0.6 x 10(5) molecules/cell), and this did not change with phorbol ester stimulation or exposure to Cd2+, arguing against a direct protective role for MT in activated neutrophils. MT mRNA levels corresponded qualitatively to expression of protein in these cells. Our data provide quantitative comparisons of leucocyte MT expression and regulation in the human population. Variation in both basal and induced MT mRNA levels reflects environmental or experimental (intra-individual) and possibly genetic (inter-individual) differences.

Cadmium↗
PubMed1989

Molecular analysis of pleckstrin: the major protein kinase C substrate of platelets.

Activation of protein kinase C (PKC) in platelets causes the immediate phosphorylation of pleckstrin, an apparent Mr 40-47,000 protein previously called 40K or P47. Pleckstrin presumably plays an important but as yet unknown role in mediating cellular responses evoked by agonist-induced phosphoinositide turnover. We have cloned the cDNA for pleckstrin from the HL-60 human promyelocytic leukemia cell line by immunological screening of a lambda gt11 expression library (Tyers et al.: Nature 333:470-473, 1988) and now report further analysis of the pleckstrin sequence. Pleckstrin has a deduced Mr of 40,087 and is encoded by a 1,050-bp open reading frame which is preceded by a short open reading frame that terminates before the correct initiator methionine. A single polymorphic site was found in the coding region. An unusual pattern of sequence heterogeneity occurred about a poly(A) tract in the 3' untranslated region. The 3.0-kb pleckstrin mRNA induced upon differentiation of HL-60 cells apparently has heterogeneous 5' ends which undergo differential regulation during HL-60 cell maturation. Analysis by multiple sequence alignment with known PKC substrates identified a strong candidate site for phosphorylation by PKC and a potential Ca2+-binding EF-hand motif. No other similarities to proteins in current databases were found.

Amino Acid Sequence↗
PubMed1989

Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen.

The complete cDNA for a human mitochondrial protein designated P1, which was previously identified as a microtubule-related protein, has been cloned and sequenced. The deduced amino acid sequence of P1 shows strong homology (40 to 50% identical residues and an additional 20% conservative replacements) to the 65-kilodalton major antigen of mycobacteria, to the GroEL protein of Escherichia coli, and to the ribulose 1,5-bisphosphate carboxylase-oxygenase (rubisco) subunit binding protein of plant chloroplasts. Similar to the case with the latter two proteins, which have been shown to act as chaperonins in the posttranslational assembly of oligomeric protein structures, it is suggested that P1 may play a similar role in mammalian cells. The observed high degree of homology between human P1 and mycobacterial antigen also suggests the possible involvement of this protein in certain autoimmune diseases.

Amino Acid Sequence↗
PubMed1988

Metallothionein RNA levels in HL-60 cells. Effect of cadmium, differentiation, and protein kinase C activation.

Metallothionein (MT) gene transcription is regulated in a developmental and tissue-specific manner by metal ions and other agents. We examined MT expression in the human promyelocytic leukemia cell line HL-60 during differentiation along macrophage and neutrophil lineages. All HL-60 phenotypes showed similar basal levels of MT RNA with significant induction following exposure to Cd2+ but not activators of PKC. MT RNA did not correlate with growth state or with known levels of PKC activity, thus our data do not support a role for MT in HL-60 differentiation or for PKC in MT expression.

Amino Acid Sequence↗
PubMed1988

Transcription initiation at the tet promoter and effect of mutations.

We have identified the startpoint for transcription in vitro of the tetracycline resistance gene (tet) of pBR322 and several deletion and insertion mutations which alter tet promoter structure. Tetracycline resistance in host bacteria correlates qualitatively with the efficiency of DNA fragments from these plasmids to promote tet transcription in vitro. Only in active promoters could we find by computer analysis promoter structures in which the -10 and -35 sequences and the relative spacing of the two regions agree with consensus sequence determinants. These data support the current model of the E. coli promoter sequence. Two promoter mutants gave heterogeneous 5' termini with additional A residues not encoded by the DNA sequence.

Base Sequence↗
PubMed1988

Molecular cloning and expression of the major protein kinase C substrate of platelets.

In platelets, agonists that stimulate phosphoinositide turnover cause the rapid phosphorylation of a protein of apparent relative molecular mass (Mr) 40-47,000, called P47, by protein kinase C (PKC). Diverse identities have been ascribed to P47 including lipocortin, inositol 1,4,5-trisphosphate 5-phosphomonoesterase, pyruvate dehydrogenase alpha subunit and an actin regulatory protein. We have isolated human P47 clones by immunological screening of a lambda gt11 complementary DNA library from HL-60 cells, a human promyelocytic leukaemia cell line. P47 recombinants thus identified hybridized to a 3.0 kilobase (kb) messenger RNA in mature white blood cell lines; the same mRNA was induced in HL-60 cells during differentiation. A 1,050 base pair (bp) open reading frame that could encode a protein of Mr40,087 was confirmed by comparison with peptide sequences from platelet P47, and by expression of the putative recombinant P47 in E. coli and in vitro. The P47 sequence appears to have been conserved throughout vertebrate evolution, and is not similar to any other known sequence including human lipocortin and the alpha subunit of pyruvate dehydrogenase. The P47 protein contains a potential Ca2+-binding 'EF-hand' structure and a region that strongly resembles known PKC phosphorylation sites.

Amino Acid Sequence↗