Biomedical subjects

C J Lloyd

Publications and source records attributed to C J Lloyd.

14 recordsLinked to original sources

PubMed1992

Alcohol and fatal road accidents: estimates of risk in Australia 1983.

This paper concerns the statistical relationship between the risk of a fatality and the age and sobriety of drivers in New South Wales, Australia. The estimation of this relationship is based on fatal accident data from New South Wales and non-accident or control data from South Australia collected between the hours of 5.00 AM and 3.00 PM. The results are presented in new ways. In particular, tables of "accidents attributable to alcohol" and age specific intensity functions of "risk attributable to alcohol" may be an aid to policy making.

Accidents, Traffic↗

PubMed1992

Chemically induced methaemoglobinaemia in a neonate.

A case of toxic methaemoglobinaemia is presented, which followed the use of a local anaesthetic containing prilocaine (Citanest, Astra Pharmaceuticals Ltd) which was used to provide analgesia prior to the suturing of a facial laceration. Although this complication is well documented, this case is unusual in that it involved a neonate and was not produced by an overdose of prilocaine (the recommendations of the British National Formulary and the product monograph were followed). Explanations for the increased susceptibility of neonates and young infants to methaemoglobinaemia are presented together with the salient clinicopathological features. The diagnostic dilemmas and investigative procedures related to the condition are considered together with the various treatments available.

Anesthesia, Local↗

PubMed1990

Detection of colonic growth factors using a human colonic carcinoma cell line (LIM1215).

Although the colonic mucosa is one of the most rapidly proliferating epithelial tissues in the body, little is known about the factors that direct this proliferation. In this report we have studied the parameters of both a mitogenic and a clonogenic assay for detecting potential colonic growth factors (CGF). Using a colon carcinoma cell line (LIM1215), which has retained a number of the properties of normal colonic mucosa, we have assayed a range of mitogenic factors for CGF activity. 3H-thymidine incorporation by the LIM1215 cell line was stimulated by low concentrations of epidermal growth factor and basic fibroblast growth factor and, to a lesser extent, by higher concentrations of interleukin-1 and insulin-like growth factor 1. The cells did not respond to a range of other mitogens and lymphokines. Optimal clonogenic response in a soft-agar assay was obtained using a primary pituitary extract.

Animals↗

PubMed1990

Inducible anti-sense RNA for angiotensinogen in stably transformed hepatoma cell lines.

Angiotensinogen mRNA is found in many extrahepatic tissues, where it may participate in local angiotensin-generating systems. In this study we explore the feasibility of using anti-sense RNA to decrease angiotensinogen production in rat H4IIEC3 hepatoma cells. An amplifiable shuttle vector was modified to allow the production of high levels of stable anti-sense RNA from two regions of the mouse angiotensinogen gene under the control of the inducible sheep metallothionein promoter. Stably transformed, clonal cell lines expressing anti-sense RNA for angiotensinogen were isolated after selection with the aminoglycoside G418. Subsequently, the number of chromosomally integrated copies of the angiotensinogen anti-sense constructs was coamplified by methotrexate selection for dihydrofolate reductase activity carried on the shuttle vector. With a 20- to 30-fold induction of the anti-sense RNAs, the target angiotensinogen mRNA level was reduced to 25-30% of control values. The specificity of this effect was confirmed by showing no decrease in either beta-tubulin or neomycin phosphotransferase mRNA levels. Using tissue-specific promoters, it should be possible to direct these effects to specific organs in transgenic mice. However, in agreement with results from other groups, our findings suggest that it will not be possible to eradicate completely the target gene product using the anti-sense RNA strategy.

Angiotensinogen↗

PubMed1988

Murine epidermal growth factor: structure and function.

Murine epidermal growth factor (EGF), a 53 amino acid protein, has been modified by enzymic digestion, site-specific chemical reactions, and recombinant DNA technology. After trypsin digestion the EGF derivatives EGF1-48 (called EGF-T) and EGF1-45 (called EGF-T2) were separated from the residual EGF and the C-terminal pentapeptide by reversed-phase high-performance liquid chromatography. EGF-T competes for binding to EGF receptors with the same efficiency as EGF. The EGF-T2 derivative had no detectable receptor binding activity even at 100 nM. The in vitro mitogenic potencies of EGF and EGF-T for Balb/c 3T3 cells were indistinguishable. Treatment of EGF-T with carboxypeptidase Y yielded two derivatives, EGF-T-(des-Arg48) and EGF-T-des(Leu47-Arg48). There was only a 3-7-fold diminution in the binding efficiency and mitogenic potency for EGF-T-(des-Arg48). However, there was more than a 100-fold decrease in the binding efficiency and mitogenic activity of EGF-T-des (Leu47-Arg48). These results indicated that Leu47 is intimately involved in the formation of the ligand-receptor complex. Studies with a number of proteases indicated that the C-terminus of EGF was susceptible to enzymic digestion; however, the N-terminus appears to be folded into a conformation which prevents access to proteolytic digestion. Consequently, the N-terminus was modified by preparing an analogue with recombinant DNA technology. Oligonucleotides corresponding to EGF(3-48). Met3 Lys21 residues were ligated in frame to a beta-galactosidase expression vector. The beta-Gal-EGF fusion protein was cleaved with cyanogen bromide and EGF(4-48).Lys21 purified.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

Doubling the one-sided P-value in testing independence in 2 x 2 tables against a two-sided alternative.

Dupont investigated the advantages of doubling the one-sided P-value in conducting a two-sided test of independence in a 2 x 2 table and claimed that the main advantage was that, unlike the 'exact' P-value, small changes in the table resulted in small changes in the significance. He admitted that this practice had no obvious theoretical justification. In this paper, I take the view that repeated sampling properties should form the basis of any such justification. I develop an appropriate framework for studying P-values and show that doubling the one-sided P-value produces a biased test whenever the table is asymmetric. I show how one may derive a slightly more complicated P-value from a uniformly most powerful test. While there are difficulties in interpreting these P-values, I argue that, in principle, one should prefer the latter. In practice the difference between the two is typically less than 10 per cent (for the tables Dupont considered, the difference is of the order of 1 per cent) but with highly skew tables the difference can be much larger.

Clinical Trials as Topic↗

PubMed1987

Primary structure of ovine pituitary basic fibroblast growth factor.

The complete amino acid sequence of basic FGF (146 residues) from ovine pituitary glands has been established. This has been achieved by the sequence analysis of subnanomole amounts of the intact molecule and of peptides derived by enzymatic digestions with clostripain, chymotrypsin, pepsin and Staphylococcus aureus V8 protease. Microbore HPLC, employing 1-2 mm i.d. columns, was used to purify, concentrate and buffer-exchange the FGF peptides. A novel application of ion-pairing chromatography was employed to isolate peptides which were not retained on conventional reversed-phase systems. There is only one positional difference between the ovine and bovine basic FGFs, but there are 3 positional differences between ovine and human basic FGFS.

Amino Acid Sequence↗

PubMed1987

Angiotensin converting enzyme induction by cyclic AMP and analogues in cultured endothelial cells.

The role of cyclic AMP in regulating the production of angiotensin converting enzyme (ACE) was investigated using cultured bovine aortic endothelial cells. Addition of dibutyryl cAMP [Bu)2cAMP) at 100 microM increased the ACE activity to 126% of control (P less than 0.005). This effect was blocked by either actinomycin D (0.1 microgram/ml) or cycloheximide (1.7 microM) indicating that RNA as well as protein synthesis was required for induction of the enzyme. After addition of (Bu)2cAMP, a lag period of 8 h was observed before increased ACE activity was detected. The stable analogues, 8-bromo cAMP (100 microM) and N6-monobutyryl cAMP (100 microM) also increased ACE activity but cAMP (100 microM) and O2-monobutyryl cAMP (100 microM) had no effect, in keeping with their susceptibility to phosphodiesterase in this system. Sodium butyrate (100 microM) was also inactive. The effect of (Bu)2cAMP on ACE was still observed in the presence of a maximal dose of dexamethasone, indicating that (Bu)2cAMP stimulates by mechanism(s) independent of the previously observed action of glucocorticoids on these cells. The phosphodiesterase inhibitor IBMX caused a dose-related increase in ACE activity with a threshold at 30 microM (P less than 0.05) and produced a 4-fold increase above control at 1 mM IBMX.

1-Methyl-3-isobutylxanthine↗

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↗