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M Boylan

Publications and source records attributed to M Boylan.

At least 19 recordsLinked to original sources
PubMed1998

The anti-proliferative effect of suramin towards tamoxifen-sensitive and resistant human breast cancer cell lines in relation to expression of receptors for epidermal growth factor and insulin-like growth factor-I: growth stimulation in the presence of tamoxifen.

BACKGROUND: A significant proportion of breast cancer patients receiving tamoxifen therapy relapse during treatment following acquisition of tamoxifen-resistant or oestrogen-independent phenotypes. The mechanism behind this rapid progression to oestrogen autonomy is at present unclear and further treatment modalities are limited. Suramin represents a novel potential second line therapy. The mechanism of the antineoplastic activity of suramin is not completely understood, although the drug binds to many growth factors including epidermal growth factor and insulin-like growth factors and can also dissociate growth factors from their receptors. In this study we have related suramin sensitivity to the expression of receptors for epidermal growth factor and insulin-like growth factor-I in a number of breast cancer cell lines including lines resistant to tamoxifen. MATERIALS AND METHODS: The anti-proliferative effects of suramin were investigated in two oestrogen dependent breast cancer lines (ZR-75-1 and MCF-7), oestrogen independent (ZR-PR-LT) and tamoxifen resistant (ZR-75-9a1) variants of ZR-75-1 and a tamoxifen resistant (LY2) variant of MCF-7. Full dose response curves were constructed and IC50 values determined for each cell line. Sensitivity to suramin was correlated with the level of expression of receptors for epidermal growth factor (EGFR) and insulin-like growth factor-I (IGFR). On observing stimulation of cell proliferation by suramin in the tamoxifen resistant cell lines in the presence of tamoxifen we also investigated the possible role of suramin sequestration of transforming growth factor-beta in mediating this effect. RESULTS: All cell lines exhibited a dose- and time-dependent response to suramin treatment. Tamoxifen resistant ZR-75-9a1 cells (day 6 IC50 85 micrograms ml-1) were more resistant to suramin than oestrogen independent ZR-PR-LT cells (day 6 IC50 45 micrograms ml-1), and the parent ZR-75-1 line (day 6 IC50 56 micrograms ml-1). Increased sensitivity to suramin was associated with increased expression of IGFR and decreased expression of EGFR. Tamoxifen resistant LY2 cells were significantly more sensitive to suramin (day 6 IC50 70 micrograms ml-1) than MCF-7 cells (day 6 IC50 350 micrograms ml-1). Both IGFR and EGFR expression by LY2 cells was lower than in the parent line. The antioestrogen-resistant ZR-75-9a1 and LY2 lines grown in the presence of 8 microM tamoxifen were growth stimulated by concentrations of the drug below 100 micrograms/ml. As growth stimulation observed in the presence of tamoxifen may have been due to suramin sequestration of tamoxifen induced TGF-beta 1 secretion we also investigated the response of the cells to this peptide in the presence and absence of suramin. All cell lines were growth inhibited by TGF-beta 1 except ZR-75-9a1 which was unresponsive. Responses to TGF-beta 1 were modified in the presence of 100 micrograms suramin ml-1 although TGF-beta 1 was unable to mimic the ability of tamoxifen to stimulate proliferation in the presence of suramin. CONCLUSIONS: These results suggest that for ZR-75-1 cells and variants, increased sensitivity to suramin is associated with an increase in expression of IGFR and a decrease in EGFR numbers. However, tamoxifen resistant LY2 cells, in which both IGFR and EGFR expression is reduced were considerably more sensitive than parental MCF-7 cells suggesting that there is no clear relationship between EGFR and IGFR expression and suramin sensitivity. The unexpected stimulation of cell proliferation of the tamoxifen resistant variants by suramin in the presence of tamoxifen could not be explained by suramin sequestration of transforming growth factor-beta and the mechanism of this interaction remains unclear.

Antineoplastic Agents↗
PubMed1996

Changes in insulin-like growth factor-I receptor expression and binding protein secretion associated with tamoxifen resistance and estrogen independence in human breast cancer cells in vitro.

Conditioned medium from a series of human breast cancer cell lines representing the phenotypes of estrogen dependence ZR-75-1, MCF-7), tamoxifen resistance (Z(+)-75-9a1, LY2) and estrogen independence (ZR-PR-Lt) contained four detectable insulin-like growth factor binding proteins (IGFBPs) of MW 24, 34, 44 and 70 kDa. in the tamoxifen resistant lines secretion of the 24 kDA IGFBP was depressed in comparison to the parent lines whilst secretion of the 44 kDa IGFBP was unaffected. Secretion of this species by ZR-PR-LT cells was reduced. Following incubation in medium containing 1% serum both tamoxifen resistant cell lines secreted higher levels of the 44 kDa IGFBP than the respective parent lines. These changes in IGFBP secretion associated with the development of tamoxifen resistance and estrogen independence were accompanied by changes in the expression of receptors for IGF-1.

Breast Neoplasms↗
PubMed1996

Expression of receptors for epidermal growth factor and insulin-like growth factor I by ZR-75-1 human breast cancer cell variants is inversely related: the effect of steroid hormones on insulin-like growth factor I receptor expression.

We have investigated the expression of insulin-like growth factor I receptors (IGFR) by the ZR-75-1 human breast cancer cell line and tamoxifen-resistant (ZR-75-9a1) and oestrogen-independent (ZR-PR-LT) variants. ZR-75-1 cells expressed 6633+/-953 receptors per cell,(K(d) 0.24+/-0.06 nM). IGFR expression was reduced in ZR-75-9a1 cells (1180+/-614 receptors per cell, K(d) 0.13+/-0.05) and increased in the ZR-PR-LT cell line (18 430+/-3210 receptors per cell, K(d) 0.24+/-17). A comparison of these data with previously published findings for epidermal growth factor receptor (EGFR) expression by these cell lines revealed that IGFR and EGFR expression are inversely related in the variant lines whereas ZR-75-1 cells express similar numbers of both receptors. Since the changes in IGFR expression observed are associated with changes in steroid hormone receptor status, we also investigated the effects of oestradiol, the synthetic progestin ORG 2058 and dexamethasone on IGFR expression. Oestradiol increased IGFR expression only in the ZR-75-1 cell line. Low concentrations of ORG 2058 increased IGFR levels in the two cell lines positive for progesterone receptor (ZR-75-1 and ZR-PR-LT). High concentrations of ORG 2058 increased IGFR expression in all cell lines, as did dexamethasone. These data suggest that EGFR and IGFR expression may be linked in breast cancer, and that EGFR/IGFR ratios in breast cancer may be a more sensitive prognostic indicator than EGFR expression alone. Regardless of basal IGFR expression by the cell studied, ORG 2058 increased IGFR expression, possibly via both the progesterone and glucocorticoid receptors.

Breast Neoplasms↗
PubMed1994

Dominant negative suppression of arabidopsis photoresponses by mutant phytochrome A sequences identifies spatially discrete regulatory domains in the photoreceptor.

We used the exaggerated short hypocotyl phenotype induced by oat phytochrome A overexpression in transgenic Arabidopsis to monitor the biological activity of mutant phytochrome A derivatives. Three different mutations, which were generated by removing 52 amino acids from the N terminus (delta N52), the entire C-terminal domain (delta C617), or amino acids 617-686 (delta 617-686) of the oat molecule, each caused striking dominant negative interference with the ability of endogenous Arabidopsis phytochrome A to inhibit hypocotyl growth in continuous far-red light ("far-red high irradiance response" conditions). By contrast, in continuous white or red light, delta N52 was as active as the unmutagenized oat phytochrome A protein in suppressing hypocotyl elongation, while delta C617 and delta 617-686 continued to exhibit dominant negative behavior under these conditions. These data suggest that at least three spatially discrete molecular domains coordinate the photoregulatory activities of phytochrome A in Arabidopsis seedlings. The first is the chromophore-bearing N-terminal domain between residues 53 and 616 that is apparently sufficient for the light-induced initiation but not the completion of productive interactions with transduction chain components. The second is the C-terminal domain between residues 617 and 1129 that is apparently necessary for completion of productive interactions under all irradiation conditions. The third is the N-terminal 52 amino acids that are apparently necessary for completion of productive interactions only under far-red high irradiance conditions and are completely dispensable under white and red light regimes.

Arabidopsis↗
PubMed1994

The SMART needle. A new Doppler ultrasound-guided vascular access needle.

Central venous access is an essential part of patient management in many clinical settings. Traditionally this has been achieved by a blind, external landmark guided technique which may not correlate exactly with the location of the vessel. We have prospectively evaluated the SMART needle, a new Doppler ultrasound guided vascular access device, in 40 patients, to evaluate whether it can improve on the standard technique. The SMART needle was easy to use and reliably distinguished between arterial and venous signals. No advantage was demonstrated in 'easy' internal jugular vein cannulations. Although ease of cannulation in difficult cases was subjectively improved, the differences in time to cannulation and number of passes between the groups failed to reach statistical significance and the complication rates were similar. However, the use of the SMART needle on two occasions enabled avoidance of carotid artery puncture by correctly distinguishing the artery from the vein, so that it may have a rôle in patients in whom difficult internal jugular venous cannulation is anticipated.

Adult↗
PubMed1992

Atheroembolism from the ascending aorta. An emerging problem in cardiac surgery.

As the ages of patients undergoing cardiac operations have increased, noncardiac causes of death have increased. To identify these causes of death, we analyzed the autopsy findings in 221 patients undergoing myocardial revascularization or valve operations between 1982 and 1989. Mean age was 65.6 +/- 9.5 years and the range was from 32 to 94 years; 130 patients (58.8%) were male. Autopsies were complete in 129 patients (58.4%) and limited to the chest and abdomen in the remainder. Embolic disease was identified in 69 patients (31.2%). Atheroemboli or abnormalities consistent with atheroemboli were identified in 48 patients (21.7%). Fourteen patients had thromboembolism and 7 had disseminated intravascular coagulation. The prevalence of atheroembolic disease increased dramatically from 4.5% in 1982 to 48.3% in 1989 (p = 0.001). Atheroembolic disease was found in the brain in 16.3% of patients, spleen in 10.9%, kidney in 10.4%, and pancreas in 6.8%. Thirty (62.5%) of the 48 patients had multiple atheroembolic sites. Atheroemboli were more common in patients undergoing coronary artery procedures (43/165; 26.1%) than in those undergoing valve procedures (5/56; 8.9%) (p = 0.008). There was a high correlation of atheroemboli with severe atherosclerosis of the ascending aorta. Atheroembolic events occurred in 46 of 123 patients (37.4%) with severe disease of the ascending aorta but in only 2 of 98 patients (2%) without significant ascending aortic disease (p less than 0.0001). Forty-six of 48 patients (95.8%) who had evidence of atheroemboli had severe atherosclerosis of the ascending aorta. There was a direct correlation between age, severe atherosclerosis of the ascending aorta, and atheroemboli. Incremental risk factors for atheroembolic are peripheral vascular disease and severe atherosclerosis of the ascending aorta.

Age Factors↗
PubMed1989

Fused bacterial luciferase subunits catalyze light emission in eukaryotes and prokaryotes.

A monocistronic luxAB gene containing the luxA and luxB genes coding for bacterial luciferase has been generated by site-specific mutagenesis so that it is now possible to express luciferase under control of a single promoter in eukaryotes as well as in prokaryotes. The fused luciferase subunits (alpha and beta), linked by a decapeptide, were synthesized in yeast under the Gal-4 promoter, in Escherichia coli under the T7-phage promoter, and in vitro in a rabbit reticulocyte lysate after transcription and capping of the mRNA under the SP6 phage promoter. Replacement of the ATG codon initiating the luxB sequence in the fused luxAB gene with CAG prevented internal initiation and allowed purification of a highly active fused luciferase in the absence of the beta-luciferase subunit. Consequently luciferase activity can be directly attributed to the fused luciferase alone and does not require complementation with free beta subunit of luciferase. Light emission could be measured in yeast and bacterial cells without the need for cell lysis providing the basis for measuring gene expression directly in vivo. These results demonstrate the potential applicability of the fused bacterial luciferase genes as a reporter of gene expression both in prokaryotic and eukaryotic systems.

Bacterial Proteins↗
PubMed1989

Lux C, D and E genes of the Vibrio fischeri luminescence operon code for the reductase, transferase, and synthetase enzymes involved in aldehyde biosynthesis.

The lux C, D, and E genes of the Vibrio fischeri luminescence operon code for three polypeptides of 54, 33, and 42 kDa, respectively, which are required for synthesis of the aldehyde substrate for the luminescent reaction. These polypeptides have been identified in V. fischeri and V. harveyi as well as in recombinant E. coli harboring the cloned genes by specific acylation with [3H]fatty acid, showing that they are components of a fatty acid reductase system with reductase, synthetase and transferase activities. By using glycerol in the assay and/or extraction buffer and decreasing the reducing agent, the levels of the acylation of the 54 and 42 kDa polypeptides have been greatly increased. As a consequence, it was possible to demonstrate that the 54 kDa polypeptide coded by the lux C gene has reductase activity. In a subclone missing the lux E gene, the 42 kDa polypeptide was missing and the 54 kDa polypeptide could not be acylated in vitro with tetradecanoic acid (+ATP) and only to a low level in vivo indicating that the synthetase enzyme, responsible for fatty acid activation, is coded by the lux E gene. In vitro acylation with tetradecanoyl CoA of the 33 kDa polypeptide coupled with the specific cleavage of acyl-ACP only in E. coli extracts transformed with DNA containing the lux D gene, demonstrated that the lux D gene coded for the transferase enzyme.

Aldehydes↗
PubMed1988

Cloning and expression of the Photobacterium phosphoreum luminescence system demonstrates a unique lux gene organization.

The organization of the lux structural genes (A-E) in Photobacterium phosphoreum has been determined and a new gene designated as luxF discovered. The P. phosphoreum luminescence system was cloned into Escherichia coli using a pBR322 vector and identified by cross-hybridization with Vibrio fischeri lux DNA. The lux genes were located by specific expression of P. phosphoreum DNA fragments in the T7-phage polymerase/promoter system in E. coli and identification of the labeled polypeptide products. The luxA and luxB gene products (luciferase subunits) were shown to catalyze light emission in the presence of FMNH2, O2, and aldehyde. The luxC, luxD, and luxE gene products (fatty acid reductase subunits) responsible for aldehyde biosynthesis could be specifically acylated with 3H-labeled fatty acids. The order of the lux genes in P. phosphoreum was found to be luxCDABFE with luxF coding for a new polypeptide of 26 kDa. The presence of a new gene in the P. phosphoreum luminescence system between luxB and luxE as compared to the organization of the lux structural gene in V. fischeri and Vibrio harveyi (luxCDABE) demonstrates that the luminescent systems in the marine bacteria have significantly diverged. The discovery of the luxF gene provides the basis for elucidating the role of its gene product in the expression of luminescence in different marine bacteria.

Cloning, Molecular↗
PubMed1988

Organization of the lux structural genes of Vibrio harveyi. Expression under the T7 bacteriophage promoter, mRNA analysis, and nucleotide sequence of the luxD gene.

The structural genes (luxA-E) of the Vibrio harveyi luminescent system coding for the luciferase (alpha, beta) and fatty acid reductase (r, s, t) polypeptides can be expressed exclusively in Escherichia coli under the T7 phage promoter providing a convenient method for identifying and locating these genes. luxD which codes for the acyltransferase enzyme producing fatty acids for the luminescent reaction was located immediately above the luciferase genes (luxA, B) with the two other fatty acid reductase genes (luxC, E) flanking these genes, in the same order as found for the Vibrio fischeri luminescent system. By hybridization with luxC DNA probes, a set of mRNAs coding for this gene was detected; part of this set of mRNAs extended downstream and complemented the set of mRNAs previously detected for the other lux structural genes. The luxD gene from a mutant (M17) which requires tetradecanoic acid for light emission was cloned into the T7 system, and upon expression it could be demonstrated that the lack of activity was due to synthesis of a full-length nonfunctional protein and not to introduction of a stop codon. The nucleotide sequence of the luxD gene of the native and mutant strains was determined and shown to consist of an open reading frame of 915 bases preceded by a Shine-Dalgarno sequence with very high homology to the ribosome binding site found for luxA and B. The 3'-sequence was identical to a 669-base open reading frame upstream of the luxA gene reported earlier by Cohn et al. (Cohn, D.H., Mileham, A.J., Simon, M.I., Nealson, K.H., Rausch, S.K., Bonam, D., and Baldwin, T.O. (1985) J. Biol. Chem. 260, 6139-6146). The luxD mutant arose by a single point mutation of G to A resulting in a change of glycine to glutamic acid.

Acylation↗
PubMed1988

A new lux gene in bioluminescent bacteria codes for a protein homologous to the bacterial luciferase subunits.

The nucleotide sequence of a new gene, luxF, located between the luxB and E genes in the bioluminescent system of Photobacterium phosphoreum has been determined. The luxF gene codes for a polypeptide of 231 amino acids which is homologous to the alpha and beta subunits of luciferase coded by the luxA and luxB genes, respectively. The degree of homology of the luxF protein is very high with the beta subunit of luciferase (approximately 30% identity) with greatest similarity to the Vibrio luxB proteins. the luxF gene appears to have evolved by duplication of the luxB gene followed by deletion of approximately 100 codons just penultimate to the 5'-terminal. The close homology with the luciferase beta subunit implicates the luxF protein in a function related to the light-emitting reaction.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of the plasmid-located determinants for CS1 and CS2 fimbriae of enterotoxigenic Escherichia coli of serotype O6:K15:H16 of human origin.

The plasmid pCS001, isolated from an enterotoxigenic strain of Escherichia coli, mediates expression of the CS1 or CS2 and CS3 fimbrial adhesins in appropriate E. coli hosts. To characterize this further, HindIII-generated DNA fragments of this plasmid were cloned into the vector plasmid pBR322. A chimaera, called pCS200, which mediated expression of the CS1 or CS2 fimbrial antigen but not of CS3 fimbrial antigen in appropriate host strains, was obtained. The DNA inserted into the vector sequences of plasmid pCS200 comprised HindIII fragments of 4.7 kbp and 0.8 kbp. Plasmid pCS200-carrying wild-type E. coli hosts of serotype O6:K15:H16 that expressed the CS1 or CS2 antigen also caused mannose-resistant agglutination of bovine red blood cells, suggesting that functional fimbriae were present on the bacterial surface. As previously observed with strain K12 recipients of CS-fimbriae-associated plasmids mobilized from wild-type enterotoxigenic E. coli, K12 recipients of the chimaeric plasmid pCS200 did not express the CS1 or CS2 fimbrial antigen. An oligonucleotide probe, synthesized on the basis of the published N-terminal amino acid sequence of the CS2 fimbrial subunit, hybridized to plasmid pCS200, indicating that the gene for the structural subunit of this fimbria resided on the plasmid.

Adhesins, Escherichia coli↗
PubMed1988

Nucleotide sequence of the gene encoding the major subunit of CS3 fimbriae of enterotoxigenic Escherichia coli.

The complete nucleotide sequence of a 612-base-pair DNA fragment containing the gene for the major fimbrial subunit of CS3 of enterotoxigenic Escherichia coli is presented. A possible promoter region, a ribosome-binding site, and two potential signal peptidase cleavage sites are indicated. Unlike the best-studied fimbrial proteins, the predicted CS3 sequence has no Cys residues.

Amino Acid Sequence↗
PubMed1987

Molecular cloning and characterization of the genetic determinant encoding CS3 fimbriae of enterotoxigenic Escherichia coli.

The genetic determinant encoding the synthesis and surface expression of CS3 fimbriae of colonization factor antigen II-(CFA/II-) positive enterotoxigenic Escherichia coli was cloned on a 5.1 kb HindIII DNA fragment in pBR322 from the wild-type plasmid pCS001 to yield the CS3+ plasmid pCS100. Subcloning of EcoRI fragments of 1.8 kb and 2.5 kb into vector plasmid pACYC184 and the isolation of a series of pCS100::Tn5 insertion mutants revealed that more than one cistron was involved in the synthesis and expression of CS3 fimbriae. Polypeptides of 94, 26, 24, 17 and 15 kDa were detected in E. coli minicells harbouring pCS100. In Western immunoblotting the 17 kDa and 15 kDa polypeptides reacted with specific anti-CS3 fimbriae serum. The 15 kDa polypeptide comigrated with the structural subunit of CS3 fimbriae. Inhibition of protein processing in minicells by ethanol confirmed that the 17 kDa polypeptide was the precursor form of the 15 kDa structural subunit. A physical map of the cloned DNA was constructed showing the location and direction of transcription of the genes for the 17 and 94 kDa polypeptides. Using the 5.1 kb HindIII fragment of pCS100 as a genetic probe for the CS3 determinant, Southern hybridization analysis of plasmid and total cellular DNA was performed in wild-type enterotoxigenic E. coli strains.

Antigens, Bacterial↗
PubMed1986

Galen

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Greek World↗
PubMed1985

Polycistronic mRNAs code for polypeptides of the Vibrio harveyi luminescence system.

DNA coding for the alpha and beta subunits of Vibrio harveyi luciferase, the luxA and luxB genes, and the adjoining chromosomal regions on both sides of these genes (total of 18 kilobase pairs) was cloned into Escherichia coli. Using labeled DNA coding for the alpha subunit as a hybridization probe, we identified a set of polycistronic mRNAs (2.6, 4, 7, and 8 kilobases) by Northern blotting; the most prominent of these was the one 4 kilobases long. This set of mRNAs was induced during the development of bioluminescence in V. harveyi. Furthermore, the same set of mRNAs was synthesized in E. coli by a recombinant plasmid that contained a 12-kilobase pair length of V. harveyi DNA and expressed the genes for the luciferase subunits. A cloned DNA segment corresponding to the major 4-kilobase mRNA coded for the alpha and beta subunits of luciferase, as well as a 32,000-dalton protein upstream from these genes that could be specifically modified by acyl-coenzyme A and is a component of the bioluminescence system. V. harveyi mRNA that was hybridized to and released from cloned DNA encompassing the luxA and luxB genes was translated in vitro. Luciferase alpha and beta subunits and the 32,000-dalton polypeptide were detected among the products, along with 42,000- and 55,000-dalton polypeptides, which are encoded downstream from the lux genes and are thought to be involved in luminescence.

Chromosome Mapping↗
PubMed1985

Functional identification of the fatty acid reductase components encoded in the luminescence operon of Vibrio fischeri.

A clone of DNA, obtained from the luminescent bacterium Vibrio fischeri ATCC 7744 and inserted into pBR322, was found to express luminescence in Escherichia coli. Polypeptides involved in biosynthesis of the fatty aldehyde substrate for the light reaction were identified by fatty acid acylation of proteins synthesized in E. coli from the recombinant plasmid. The cloned region was similar to that reported for the V. fischeri MJ1 luminescence system (Engebrecht et al., Cell 32:773-781), except for some differences in endonuclease restriction sites and the requirement of a lower temperature for the expression of light in our cloned system. Fatty acid reductase activity could be detected in extracts of E. coli harboring the recombinant plasmid but not in extracts of the parental V. fischeri strain. Using in vivo labeling with [3H]tetradecanoic acid, we showed that the acylated polypeptides synthesized in the cloned system corresponded to the labeled polypeptides in V. fischeri (34, 42, and 54 kilodaltons) and that they could only be detected after induction of luminescence. These results provide direct evidence that the genes coding for the fatty acid reductase polypeptides are an integral part of the luminescence operon in the V. fischeri luminescence system.

Acylation↗