Biomedical subjects

C J Lloyd

Publications and source records attributed to C J Lloyd.

24 records · Page 2Linked to original sources

PubMed1986

Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.

A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub-populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry.

Carcinoma, Squamous Cell↗

PubMed1985

Rat epidermal growth factor: complete amino acid sequence. Homology with the corresponding murine and human proteins; isolation of a form truncated at both ends with full in vitro biological activity.

Epidermal growth factor (EGF) isolated from the submaxillary gland of the rat (rEGF) is missing the COOH-terminal five residues present in both mouse and human EGF. rEGF competes for the binding of 125I-labelled mEGF to human carcinoma cells with the same affinity as mEGF. rEGF and mEGF have identical mitogenic activities on mouse 3T3 fibroblasts, thus the C-terminal region of the sequence is not necessary for the in vitro activity of EGF. Using reversed-phase high-performance liquid chromatography, four molecular forms of EGF have been extracted from rat submaxillary glands. These forms represent rEGF, rEGF(2-48), rEGF(3-48) and rEGF(4-48); all forms appear to be equipotent in both the receptor binding and mitogenic assays. The isoelectric points of these rEGFs are in the range of pH 5.1 to 5.2. The primary structure of rEGF was determined from approximately 10 micrograms protein by sequence analysis of the intact molecule and fragments obtained from the reduced and alkylated protein by chemical cleavage with CNBr and enzymic cleavage with chymotrypsin and a proline-specific endopeptidase. Subnanomole amounts of generated peptides were purified to homogeneity by reversed-phase microbore high-performance liquid chromatography and analysed by automated Edman degradation in a gas-phase sequencer. There are 48 amino acid residues in the complete polypeptide chain which lacks alanine, phenylalanine, lysine and tryptophan. The amino acid sequence of rat epidermal growth factor is: Asn-Ser-Asn-Thr-Gly-Cys-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-Cys-Leu-Asn- Gly-Gly-Val-Cys-Met-Tyr-Val-Glu-Ser-Val-Asp-Arg-Tyr-Val-Cys-Asn-Cys -Val-Ile-Gly-Tyr-Ile-Gly-Glu-Arg-Cys-Gln-His-Arg-Asp-Leu-Arg. The calculated relative molecular mass from the sequence analysis is 5377.

Amino Acid Sequence↗

PubMed1983

Murine epidermal growth factor: heterogeneity on high resolution ion-exchange chromatography.

We have shown that epidermal growth factor (EGF) purified either by the classical method of Savage and Cohen, or solely by h.p.l.c. techniques can be resolved into two species, EGF alpha and EGF beta. However, despite the apparent purity of such materials, as determined both chromatographically and by amino acid analysis, they failed to give homogeneous products on radioiodination. Analysis by isoelectric focusing on agarose gels followed by transfer to nitrocellulose and silver staining showed that EGF alpha could be further resolved into three sub-species which focused at pH 4.6, 4.3 and 4.1. EGF beta (which also focused at pH 4.6) contained very small amounts of the species with isoelectric points of 4.1 and 4.3, probably due to slight contamination of this preparation by EGF alpha. Preparative separation of the sub-species of EGF alpha was achieved by high performance anion-exchange chromatography at pH 6.5 on a Pharmacia Mono Q column. Radioiodination of these purified sub-species did not produce significant charge heterogeneity. However, two slightly different forms of [125I]EGF alpha 1 (pH 4.6 species) were separable by anion-exchange chromatography on the Mono Q column. All of the EGF species competed for binding to EGF receptors on A431 cells and were active mitogens for BALB/c 3T3 fibroblasts.

Animals↗

PubMed1983

Glucocorticoid-induced proteins in bovine endothelial cells.

High circulating levels of corticosteroids are associated with elevated blood pressure and an increased incidence of vascular damage. We have used high resolution two-dimensional gel electrophoresis and autoradiography to analyse direct steroid effects on the newly synthesized proteins in bovine aortic endothelial cells. At medium concentrations of 10(-7) M, corticosteroids but not sex steroids increased the synthesis of two endothelial proteins: el (M.W. approximately 43K, pI approximately 6.3) and e2 (M.W. approximately 28K, pI approximately 5.9). The responses were consistent for different endothelial cell cultures, different gel runs, and for natural and synthetic glucocorticoids, including the 'pure' synthetic glucocorticoid, RU 26988. Mineralocorticoids (aldosterone, deoxycorticosterone) at 10(-7) M were less potent in their effect on the synthesis of e2, and had no discernible effect on the synthesis of e1. These specific effects on cellular protein synthesis demonstrate that glucocorticoids can act directly on endothelial cells. These direct actions may thus mediate altered endothelial cell function in clinical and experimental cardiovascular conditions characterized by corticosteroid excess.

Aldosterone↗

PubMed1982

Induction by glucocorticoids of angiotensin converting enzyme production from bovine endothelial cells in culture and rat lung in vivo.

The effect of corticosteroids on angiotensin converting enzyme was investigated in endothelial cell cultures and intact rat lung. Cultured endothelial cells from bovine aorta showed net production of angiotensin converting enzyme (ACE) over 2 d culture in serum-free medium. Dexamethasone (DM) increased cell ACE activity six- to sevenfold at 100 nM with a threshold effect at 0.3 nM. The effect of DM on ACE production was completely inhibited by actinomycin D or cycloheximide. Deoxycorticosterone (DOC) and aldosterone were markedly less active, with a threshold near 100 nM and significant (two to threefold) stimulation of ACE activity at 1 muM. In cells incubated in the presence of 10 nM DM, DOC (10 muM) significantly inhibited ACE production compared with 10 nM DM alone, suggesting that DOC is a partial agonist/partial antagonist in this enzyme system. Protein content of cells or medium was unchanged by steroids at all doses used. In vivo, adrenalectomized rats showed lower pulmonary ACE compared with intact controls, and when injected with DM (40 mug/d for 4 d) showed a significant (twofold, P < 0.002) increase in lung ACE over oil-injected, adrenalectomized controls; serum ACE did not change. Injection with DOC (40 mug/d) or aldosterone (10 mug/d) had no effect on lung or serum ACE. Over a range (0.6 to 2,000 mug) of concentrations of DM administered daily for 7 d, the dose-response curve of DM for induction of pulmonary ACE mirrored that for thymolysis; for both, half-maximal effects were seen at approximately 6 mug DM/d, and plateau levels at 60 mug/d. We conclude that glucocorticoids are potent inducers of ACE activity in endothelial cells in culture and in rat lung in vivo, and that the action of aldosterone and DOC reflects occupancy of glucocorticoid receptors. This effect may be of (patho)physiological relevance in regulating levels of ACE in local vascular beds, and thereby modulating local levels of the vasoactive peptides angiotensin II and bradykinin.

Animals↗

PubMed1982

Glucocorticoid induction of angiotensin converting enzyme production from bovine endothelial cells in culture and rat lung in vivo.

1. Endothelial cells from bovine aortae, grown to confluence in culture and then maintained for 2 days in serum-free medium produced an enzyme which closely resembled angiotensin converting enzyme (ACE). 2. Dexamethasone increased cell ACE activity 6- to 7-fold with a threshold near 0.3 nmol/l. This effect was completely inhibited by actinomycin D or cycloheximide. 3. Deoxycorticosterone (DOC) and aldosterone were approximately 0.1% as active as dexamethasone. 4. In cells incubated in the presence of dexamethasone (10 nmol/l), DOC (10 mumol/l) inhibited ACE production suggesting that DOC is a partial glucocorticoid agonist/antagonist in this system. 5. Adrenalectomized rats had lower pulmonary ACE compared to intact controls. This was restored by injections of dexamethasone (40 micrograms/day) but not by DOC (40 micrograms/day) or aldosterone (10 micrograms/day). The dose response curve for dexamethasone induction of pulmonary ACE mirrored that for thymolysis; for both, half maximal effects were seen at approximately equal to 6 micrograms/day and plateau levels at approximately equal to 60 micrograms/day. 6. Glucocorticoids appear to be potent inducers of ACE activity in endothelial cells in culture and rat lung in vivo. The action of DOC and aldosterone probably reflects occupancy of glucocorticoid receptors. This effect may be of significance in modulating ACE in local vascular beds.

Animals↗