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M I Bird

Publications and source records attributed to M I Bird.

36 records · Page 2Linked to original sources
PubMed1996

Role for L-selectin in lipopolysaccharide-induced activation of neutrophils.

The activation of leucocytes by bacterial cell wall lipopolysaccharide (LPS) contributes to the pathogenesis of septic shock. LPS is known to interact with several cell-surface proteins, including CD14, when presented as a complex with serum LPS-binding protein. However, the identity of the receptor responsible for LPS signalling and leucocyte activation is unknown. Interestingly, mice deficient in cell-surface L-selectin were dramatically resistant to the lethal effects of high doses of LPS in a model of septic shock. Recently we reported that L-selectin binds to cardiolipin and other charged phospholipids at a site distinct from the carbohydrate-binding site. Structural similarities between charged phospholipids and the lipid A moiety of LPS prompted us to investigate interactions between L-selectin and LPS. Herein we show that L-selectin is a neutrophil surface receptor for LPS and lipotechoic acid. The binding of LPS to L-selectin is independent of serum and Ca2+, and is blocked by antibodies to L-selectin and fucoidan. Furthermore, the interaction of LPS with cell-surface L-selectin results in superoxide production, indicating that L-selectin can mediate both binding and activation of human neutrophils. These findings suggest novel therapeutic approaches for the treatment of septic shock.

Animals↗
PubMed1996

Anionic phospholipids bind to L-selectin (but not E-selectin) at a site distinct from the carbohydrate-binding site.

It is known that L-selectin binds to glycoconjugates containing the tetrasaccharide sialyl Lewis X in a Ca2+-dependent manner. In addition, a number of other acidic oligosaccharides (for example heparin or chondroitin sulphate) or glycolipids (for example sulphatides) bind to L-selectin independent of cations. In this paper we have established that L-selectin binds to charged phospholipids, such as cardiolipin and phosphatidylserine, but not to neutral phospholipids such as phosphatidylcholine. No interaction between E-selectin and any phospholipid was observed. The interaction between L-selectin cardiolipin was inhibited by dextran sulphate, fucoidan, mannose 6-phosphate and monoclonal antibodies previously reported to block the interaction between L-selectin and its natural ligands. Analysis of the amino acid sequence of the selectins indicated that L-selectin, but no E-selectin, contains a sequence homologous to the putative cardiolipin-binding epitope found in plasma glycoprotein beta2I. Glycoprotein beta2I and a peptide corresponding to the putative cardiolipin-binding epitope in beta2I inhibited the binding of L-selectin to cardiolipin or fucoidin. Based on the binding characteristics, sequence analysis and structural modelling of L-selectin, we suggest that the amino acid sequence KKNKED (residues 84-89) is a novel site for the binding of acidic species to L-selectin. This motif is localized close to the putative carbohydrate-binding domain of L-selectin and may be a second site within the lectin domain for the interaction of leucocyte L-selectin with its natural endothelial ligands.

Amino Acid Sequence↗
PubMed1991

Two nonradioactive assays for phosphotyrosine phosphatases with activity toward the insulin receptor.

Two highly sensitive, nonradiolabeled assays for protein phosphotyrosine phosphatase (PTPase) have been developed. The first assay is based on the use of chemically synthesised phosphotyrosine-containing peptides that can be separated from the dephosphorylated peptide products by HPLC. In this assay, partially purified placental PTPase 1B dephosphorylated three dodecaphosphopeptides (corresponding to insulin receptor autophosphorylation sites at positions PY1146, PY1150, and PY1151) with approximately equal affinity (Km 1.3-2.5 microM), indicating that PTPase 1B shows no distinct preference for the site of dephosphorylation in these peptides. The second assay employs either a phosphopeptide or an autophosphorylated tyrosine kinase domain immobolized on microtiter plate wells. After reaction with PTPase, the remaining unconverted phosphosubstrate is detected in an ELISA using anti-phosphotyrosine antibodies. The latter assay was used to monitor PTPase activity during purification procedures and for characterizing PTPases. Modulation of PTPase activity by orthovanadate, heparin, Zn2+, and EDTA gave similar results in both assays. The immobilized autophosphorylated IR tyrosine kinase domain was a poor substrate for bovine liver alkaline phosphatase and seminal fluid acid phosphatase. The second assay also offers the potential for comparing PTPase activity toward several autophosphorylated tyrosine kinase domains, including those of the insulin, epidermal growth factor, and platelet-derived growth factor receptors.

Amino Acid Sequence↗
PubMed1989

Feeding pure docosahexaenoate or arachidonate decreases plasma triacylglycerol secretion in rats.

Essential fatty acid (EFA)-deficient rats were fed highly purified methyl esters of docosahexaenoate (22:6n-3), arachidonate (20:4n-6), alpha-linolenate (18:3n-3) or oleate (18:1n-9) (100 mg/day, tube fed for 3-10 days), and their plasma triacylglycerol (TG) secretion rates were measured. Secretion rates of TG into plasma were reduced by tube-feeding 22:6n-3, 20:4n-6, 18:3n-3, but not 18:1n-9, to EFA-deficient rats. A significant reduction occurred after feeding 22:6n-3 for only three days. Feeding 22:6n-3 or 18:3n-3 to EFA-deficient rats for three days also reduced the activities of liver lipogenic enzymes and sharply increased the proportions of 22:6n-3 and 20:5n-3 in liver phospholipid fractions. Mechanisms by which these EFA may reduce lipogenesis are discussed.

ATP Citrate (pro-S)-Lyase↗
PubMed1987

Enzyme activities in regions of the hypothalamus.

The activities of certain key enzymes have been measured in the ventral medial and ventral lateral areas of the hypothalamus, which are implicated in feeding behaviour, and compared with enzyme activities in the cortex and brainstem. The enzymes measured are concerned with glucose metabolism [hexokinase (EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49)], ketone body metabolism [3-hydroxybutyrate dehydrogenase (EC 1.1.1.30)], fatty acid utilisation [carnitine palmitoyl transferase (EC 2.3.1.7)], citric acid cycle activity [pyruvate dehydrogenase (EC 1.2.4.2) and citrate synthase (EC 4.1.3.7)] and neurotransmitter synthesis [glutamate dehydrogenase (EC 1.4.1.3)].

Animals↗
PubMed1985

Interacting effects of L-carnitine and malonyl-CoA on rat liver carnitine palmitoyltransferase.

Malonyl-CoA significantly increased the Km for L-carnitine of overt carnitine palmitoyltransferase in liver mitochondria from fed rats. This effect was observed when the molar palmitoyl-CoA/albumin concentration ratio was low (0.125-1.0), but not when it was higher (2.0). In the absence of malonyl-CoA, the Km for L-carnitine increased with increasing palmitoyl-CoA/albumin ratios. Malonyl-CoA did not increase the Km for L-carnitine in liver mitochondria from 24h-starved rats or in heart mitochondria from fed animals. The Km for L-carnitine of the latent form of carnitine palmitoyltransferase was 3-4 times that for the overt form of the enzyme. At low ratios of palmitoyl-CoA/albumin (0.5), the concentration of malonyl-CoA causing a 50% inhibition of overt carnitine palmitoyltransferase activity was decreased by 30% when assays with liver mitochondria from fed rats were performed at 100 microM-instead of 400 microM-carnitine. Such a decrease was not observed with liver mitochondria from starved animals. L-Carnitine displaced [14C]malonyl-CoA from liver mitochondrial binding sites. D-Carnitine was without effect. L-Carnitine did not displace [14C]malonyl-CoA from heart mitochondria. It is concluded that, under appropriate conditions, malonyl-CoA may decrease the effectiveness of L-carnitine as a substrate for the enzyme and that L-carnitine may decrease the effectiveness of malonyl-CoA to regulate the enzyme.

Acyl Coenzyme A↗
PubMed1985

Effects of DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA in rat liver and heart mitochondria. Inhibition of carnitine palmitoyltransferase and displacement of [14C]malonyl-CoA from mitochondrial binding sites.

The overt form of carnitine palmitoyltransferase (CPT1) in rat liver and heart mitochondria was inhibited by DL-2-bromopalmitoyl-CoA and bromoacetyl-CoA. S-Methanesulphonyl-CoA inhibited liver CPT1. The inhibitory potency of DL-2-bromopalmitoyl-CoA was 17 times greater with liver than with heart CPT1. Inhibition of CPT1 by DL-2-bromopalmitoyl-CoA was unaffected by 5,5'-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. In experiments in which DL-2-bromopalmitoyl-CoA displaced [14C]malonyl-CoA bound to liver mitochondria, the KD (competing) was 25 times the IC50 for inhibition of CPT1 providing evidence that the malonyl-CoA-binding site is unlikely to be the same as the acyl-CoA substrate site. Bromoacetyl-CoA inhibition of CPT1 was more potent in heart than in liver mitochondria and was diminished by 5,5'-dithiobis-(2-nitrobenzoic acid) or (in liver) by starvation. Bromoacetyl-CoA displaced bound [14C]malonyl-CoA from heart and liver mitochondria. In heart mitochondria this displacement was competitive with malonyl-CoA and was considerably facilitated by L-carnitine. In liver mitochondria this synergism between carnitine and bromoacetyl-CoA was not observed. It is suggested that bromoacetyl-CoA interacts with the malonyl-CoA-binding site of CPT1. L-Carnitine also facilitated the displacement by DL-2-bromopalmitoyl-CoA of [14C]malonyl-CoA from heart, but not from liver, mitochondria. DL-2-Bromopalmitoyl-CoA and bromoacetyl-CoA also inhibited overt carnitine octanoyl-transferase in liver and heart mitochondria. These findings are discussed in relation to inter-tissue differences in (a) the response of CPT1 activity to various inhibitors and (b) the relationship between high-affinity malonyl-CoA-binding sites and those sites for binding of L-carnitine and acyl-CoA substrates.

Acetyl Coenzyme A↗
PubMed1985

Carnitine acyltransferase activities in rat brain mitochondria. Bimodal distribution, kinetic constants, regulation by malonyl-CoA and developmental pattern.

Carnitine palmitoyltransferase and carnitine octanoyltransferase activities in brain mitochondrial fractions were approx. 3-4-fold lower than activities in liver. Estimated Km values of CPT1 and CPT2 (the overt and latent forms respectively of carnitine palmitoyltransferase) for L-carnitine were 80 microM and 326 microM, respectively, and K0.5 values for palmitoyl-CoA were 18.5 microM and 12 microM respectively. CPT1 activity was strongly inhibited by malonyl-CoA, with I50 values (concn. giving 50% of maximum inhibition) of approx. 1.5 microM. In the absence of other ligands, [2-14C]malonyl-CoA bound to intact brain mitochondria in a manner consistent with the presence of two independent classes of binding sites. Estimated values for KD(1), KD(2), N1 and N2 were 18 nM, 27 microM, 1.3 pmol/mg of protein and 168 pmol/mg of protein respectively. Neither CPT1 activity, nor its sensitivity towards malonyl-CoA, was affected by 72 h starvation. Rates of oxidation of palmitoyl-CoA (in the presence of L-carnitine) or of palmitoylcarnitine by non-synaptic mitochondria were extremely low, indicating that neither CPT1 nor CPT2 was likely to be rate-limiting for beta-oxidation in brain. CPT1 activity relative to mitochondrial protein increased slightly from birth to weaning (20 days) and thereafter decreased by approx. 50%.

Acyltransferases↗
PubMed1984

Formation of glycine and aminoacetone from L-threonine by rat liver mitochondria.

Threonine is a precursor of glycine in the rat, but the metabolic pathway involved is unclear. To elucidate this pathway, the biosynthesis of glycine, and of aminoacetone, from L-threonine were studied in rat liver mitochondrial preparations of differing integrities. In the absence of added cofactors, intact mitochondria formed glycine and aminoacetone in approximately equal amounts from 20 mM L-threonine, but exogenous NAD+ decreased and CoA increased the ratio of glycine to aminoacetone formed. In intact and freeze-thawed mitochondria, the ratio of glycine to aminoacetone formed was markedly sensitive to the concentration of L-threonine, glycine being the major product at low L-threonine concentrations. Disruption of mitochondrial integrity by sonication (1 min) decreased the ratio of glycine to aminoacetone formed, and in 20000 X g supernatant fractions from sonicated (3 min) mitochondria, aminoacetone was the major product. The main non-nitogenous two-carbon compound detected when intact mitochondria catabolized L-threonine to glycine was acetate, which was probably derived from deacylation of acetyl-CoA. These results suggest that glycine formation from L-threonine in rat liver mitochondria occurred primarily by the coupled activities of threonine dehydrogenase and 2-amino-3-oxobutyrate CoA-ligase, the extent of coupling between the enzymes being dependent upon a close physical relationship and upon the flux through the dehydrogenase reaction. In vivo glycine synthesis would predominate, and aminoacetone would be a minor product.

Acetone↗
PubMed1984

Cycloheximide blocks changes in rat liver carnitine palmitoyltransferase 1 activity in starvation.

Starvation (24h) increased the maximum activity of carnitine palmitoyltransferase 1 in rat liver and increased the concentration of malonyl-CoA required to cause 50% inhibition of the enzyme (I50). Re-feeding (24h) with a standard cube diet or a high-carbohydrate diet reversed both of these changes, whereas re-feeding with a high-fat diet did not. Administration of cycloheximide (200 micrograms/100 g body wt.) blocked the increases in carnitine palmitoyltransferase 1 activity and I50 on starvation. It is suggested that increase in carnitine palmitoyltransferase 1 activity in starvation may involve synthesis of new enzyme.

Acyltransferases↗
PubMed1984

Binding of malonyl-CoA to isolated mitochondria. Evidence for high- and low-affinity sites in liver and heart and relationship to inhibition of carnitine palmitoyltransferase activity.

[14C]Malonyl-CoA bound to intact mitochondria isolated from rat liver and heart in a manner consistent with the presence of two independent classes of binding sites in each tissue. The binding characteristics for mitochondria obtained from fed male rats were: for heart, KD(1) = 11-18nM, KD(2) = 30 microM, N1 = 7pmol/mg of protein, N2 = approx. 660pmol/mg of protein; for liver, KD(1) = 0.1 microM, KD(2) = 5.6 microM, N1 = 11pmol/mg of protein, N2 = 165pmol/mg of protein. In the presence of 40 microM-palmitoyl-CoA the characteristics of binding at the high-affinity sites were changed, so that for heart KD(1) = 0.26 microM, with no change in N1 and for liver KD(1) = approx. 2 microM, with N1 increased to approx. 40pmol/mg of protein. Differences between the two tissues in tightness of malonyl-CoA binding at the high-affinity sites explains the considerably greater sensitivity of heart CPT1 (overt form of carnitine palmitoyltransferase) to inhibition by malonyl-CoA [Saggerson & Carpenter, (1981) FEBS Lett. 129, 229-232; McGarry, Mills, Long & Foster (1983) Biochem. J. 214, 21-28]. Starvation (24h) did not change the characteristics of [14C]malonyl-CoA binding to liver mitochondria and did not alter the I50 (concentration giving 50% inhibition) for displacement of [14C]malonyl-CoA by palmitoyl-CoA. Therefore the decreased sensitivity of liver CPT1 to inhibition by malonyl-CoA in starvation [Saggerson & Carpenter (1981) FEBS Lett. 129, 225-228; Bremer (1981) Biochim. Biophys. Acta 665, 628-631] is not explained by differences in malonyl-CoA binding. Percentage occupancy of the high-affinity sites in heart mitochondria by malonyl-CoA correlated closely with percentage inhibition of CPT1 measured under similar conditions. This finding supports the proposal that the high-affinity binding sites are the functional sites mediating inhibition of CPT1 by malonyl-CoA. Similar experiments with liver mitochondria also suggested that the occupancy of high-affinity sites by malonyl-CoA regulates CPT1 activity. 5,5'-Dithiobis-(2-nitrobenzoic acid), which decreased the sensitivity of heart or liver CPT1 to inhibition by malonyl-CoA [Saggerson & Carpenter (1982) FEBS Lett. 137, 124-128], also decreased [14C]malonyl-CoA binding to the high-affinity sites of heart mitochondria. N1 values for [14C]malonyl-CoA binding to high-affinity sites in liver mitochondria were determined in various physiological states which encompassed a 7-fold range of CPT1 maximal activity (fed, starved, pregnant, hypothyroid, foetal). The N1 value did not change in these states.(ABSTRACT TRUNCATED AT 400 WORDS)

Acyl Coenzyme A↗
PubMed1983

Metabolic homoeostasis of L-threonine in the normally-fed rat. Importance of liver threonine dehydrogenase activity.

Threonine dehydratase, threonine aldolase and threonine dehydrogenase activities were assayed in livers of rats that had been normally-fed, starved for 72 h, fed a high-protein diet or normally-fed and injected with glucagon or cortisone. A modified continuous spectrophotometric assay for threonine aldolase overcame interference resulting from threonine dehydratase activity and revealed that threonine aldolase activity was very low in rat liver, irrespective of the metabolic state of the animal. The concentration of free threonine was determined in livers of animals subjected to the same treatments as described above. Using Michaelis-Menten kinetics to estimate enzyme activities in vivo at intracellular threonine concentrations it was calculated that in the normally-fed state, 87% of the threonine degraded was catabolized by threonine dehydrogenase. In other metabolic states (except in glucagon-treated animals) threonine dehydratase was the major enzyme catalysing threonine catabolism. It was concluded that threonine dehydrogenase activity plays a hitherto unrecognized role in the metabolic homoeostasis of threonine in the normally-fed rat and that this enzyme activity, in association with 2-amino-3-oxobutyrate CoA-ligase, accounts for the known rate of glycine formation from threonine in the rat.

Alcohol Oxidoreductases↗
PubMed1982

Triacylglycerol secretion in rats: effects of essential fatty acids and influence of dietary sucrose, glucose or fructose.

The rate of triacylglycerol (TG) secretion into plasma was determined in vivo in essential-fatty-acid(EFA)-adequate and EFA-deficient rats fed for 8-10 weeks on diets containing either sucrose, glucose or fructose (66.5% wt/wt) as the sole source of carbohydrate. The rate of TG secretion was increased in EFA-deficient rats, irrespective of the type of dietary carbohydrate. The secretion rate in EFA-deficient rats fed fructose was 50% higher than the rate in similar animals fed glucose or sucrose, although in the EFA-adequate rats, the secretion rate with fructose was not significantly higher than with sucrose or glucose. The concentrations of triacylglycerols and cholesteryl esters in plasma were generally reduced in EFA deficiency, whereas the concentrations of these lipids in liver were increased. Plasma free fatty acid concentrations were unchanged. The fatty acid composition of plasma and hepatic lipids was altered both by EFA deficiency and to a smaller extent by the type of dietary carbohydrate. The concentration of plasma glucagon was reduced in the deficient animals, although plasma insulin concentration was not affected. The resulting increase in the plasma insulin:glucagon molar ratio may explain some of the alterations in lipid concentrations and in lipid metabolism observed in EFA deficiency.

Animals↗