Mechanistic alternatives in phosphate monoester hydrolysis: what conclusions can be drawn from available experimental data?
(1/2031)
Phosphate monoester hydrolysis reactions in enzymes and solution are often discussed in terms of whether the reaction pathway is associative or dissociative. Although experimental results for solution reactions have usually been considered as evidence for the second alternative, a closer thermodynamic analysis of observed linear free energy relationships shows that experimental information is consistent with the associative, concerted and dissociative alternatives. (+info)
Effects of 2 low-fat stanol ester-containing margarines on serum cholesterol concentrations as part of a low-fat diet in hypercholesterolemic subjects.
(2/2031)
BACKGROUND: Full-fat sitostanol ester-containing margarine reduces serum total and LDL cholesterol, but the effect of plant stanol ester-containing margarine as part of a low-fat, low-cholesterol diet has not been studied. OBJECTIVE: We investigated the cholesterol-lowering effects of 2 novel, low-fat stanol ester-containing margarines as part of a low-fat diet recommended for hypercholesterolemic subjects. DESIGN: In a parallel, double-blind study, 55 hypercholesterolemic subjects were randomly assigned after a 4-wk high-fat diet (baseline) to 3 low-fat margarine groups: wood stanol ester-containing margarine (WSEM), vegetable oil stanol ester-containing margarine (VOSEM), and control margarine (no stanol esters). The groups consumed the margarines for 8 wk as part of a diet resembling that of the National Cholesterol Education Program's Step II diet. The daily mean total stanol intake was 2.31 and 2.16 g in the WSEM and VOSEM groups, respectively. RESULTS: During the experimental period, the reduction in serum total cholesterol was 10.6% (P < 0.001) and 8.1% (P < 0.05) greater and in LDL cholesterol was 13.7% (P < 0.01) and 8.6% (P = 0.072) greater in the WSEM and VOSEM groups, respectively, than in the control group. Serum campesterol concentrations decreased 34.5% and 41.3% (P < 0.001) in the WSEM and VOSEM groups, respectively. Serum HDL cholesterol, sitostanol, campestanol, beta-carotene, and fat-soluble vitamin concentrations did not change significantly from baseline. CONCLUSIONS: We conclude that the low-fat, plant stanol ester-containing margarines are effective cholesterol-lowering products in hypercholesterolemic subjects when used as part of a low-fat, low-cholesterol diet. They offer an additional, clinically significant reduction in serum cholesterol concentrations to that obtained with a low-fat diet alone. (+info)
High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2).
(3/2031)
Binding of fluorescent fatty acids to bovine liver non-specific lipid-transfer protein (nsL-TP) was assessed by measuring fluorescence resonance energy transfer (FRET) between the single tryptophan residue of nsL-TP and the fluorophore. Upon addition of pyrene dodecanoic acid (Pyr-C12) and cis-parinaric acid to nsL-TP, FRET was observed indicating that these fatty acids were accommodated in the lipid binding site closely positioned to the tryptophan residue. Substantial binding was observed only when these fatty acids were presented in the monomeric form complexed to beta-cyclodextrin. As shown by time-resolved fluorescence measurements, translocation of Pyr-C12 from the Pyr-C12-beta-cyclodextrin complex to nsL-TP changed dramatically the direct molecular environment of the pyrene moiety: i.e. the fluorescence lifetime of the directly excited pyrene increased at least by 25% and a distinct rotational correlation time of 7 ns was observed. In order to evaluate the affinity of nsL-TP for intermediates of the beta-oxidation pathway, a binding assay was developed based on the ability of fatty acyl derivatives to displace Pyr-C12 from the lipid binding site as reflected by the reduction of FRET. Hexadecanoyl-CoA and 2-hexadecenoyl-CoA were found to bind readily to nsL-TP, whereas 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA bound poorly. The highest affinities were observed for the very-long-chain fatty acyl-CoA esters (24:0-CoA, 26:0-CoA) and their enoyl derivatives (24:1-CoA, 26:1-CoA). Binding of non-esterified hexadecanoic acid and tetracosanoic acid (24:0) was negligible. (+info)
Preparation of isolated intestinal villi useful for studying hydrolysis rates of penicillin and cephalosporin esters.
(4/2031)
A method for the preparation of freeze-dried intestinal villi from hamsters and mice is described. The villi, consisting largely of epithelial cells rich in esterase activity, are useful for hydrolysis studies of penicillin and cephalosporin esters. (+info)
Amplification of the antibody response by C3b complexed to antigen through an ester link.
(5/2031)
Complement C3 has been described as playing an important role in the cell-mediated immune response. C3b has the capacity to covalently bind Ag and then to stimulate in vitro Ag presentation to T lymphocytes. To verify this observation in vivo, we prepared and purified covalent human C3b-Ag complexes using lysozyme (HEL) as Ag. The characterization of these HEL-C3b complexes indicates that they are representative of those susceptible to be generated in physiological conditions. Mice were immunized with 0.1 to 0.6 microgram of either free HEL, HEL + C3b, HEL-C3b, or HEL + CFA. Response was assessed after two i.p. injections by quantification of specific Ab production. Immunization with either HEL-C3b complexes or HEL + CFA leads to anti-HEL IgG production whereas free HEL or HEL + C3b was ineffective. Either HEL-C3b or HEL + CFA immunizations led to a similar Ig subclass patterns, including IgG1, IgG2a, IgA, and IgM. Our experiments provide the first evidence for modulation of specific Ab response by C3b when it is bound to Ag through a physiological-like link. Taken together with previous data concerning Ab response following recombinant HEL-C3d immunization, cellular events such as processing of C3b-Ag by APC and recognition by T lymphocytes, this present result underlines the importance of C3b and its fragments in stimulation of the immune system, through the multiplicity and complementarity of its interactions. (+info)
Intermediates of myocardial mitochondrial beta-oxidation: possible channelling of NADH and of CoA esters.
(6/2031)
Adult rat heart mitochondria were isolated and incubated with [U-14C]hexadecanoyl-CoA or unlabelled hexadecanoyl-CoA. The accumulating CoA and carnitine esters and [NAD+]/[NADH] ratio were measured by HPLC or tandem mass spectrometry. Despite minimal changes in the intramitochondrial [NAD+]/[NADH] ratio, 2, 3-unsaturated and 3-hydroxyacyl esters were observed as well as saturated acyl-CoA and acylcarnitine esters. In addition to acetylcarnitine, significant amounts of butyryl-, hexanoyl-, octanoyl- and decanoylcarnitines were detected and measured. Rat myocardial beta-oxidation is subject to control at the level of 3-hydroxyacyl-CoA dehydrogenase but this control is not due to a simple lack of oxidised NAD. We hypothesise a pool of NAD in contact between the trifunctional protein of beta-oxidation and complex I of the respiratory chain, the turnover of which is responsible for some of the control of beta-oxidation flux. In addition, short- and medium-chain acylcarnitine esters were detected whereas only small amounts of long-chain acylcarnitines were present. This may imply the presence of a mitochondrial carnitine octanoyl transferase or may reflect channelling of long-chain CoA esters so that they are not available for carnitine palmitoyl transferase II activity. (+info)
Serum sterols during stanol ester feeding in a mildly hypercholesterolemic population.
(7/2031)
We investigated the changes of cholesterol and non-cholesterol sterol metabolism during plant stanol ester margarine feeding in 153 hypercholesterolemic subjects. Rapeseed oil (canola oil) margarine without (n = 51) and with (n = 102) stanol (2 or 3 g/day) ester was used for 1 year. Serum sterols were analyzed with gas-liquid chromatography. The latter showed a small increase in sitostanol peak during stanol ester margarine eating. Cholestanol, campesterol, and sitosterol proportions to cholesterol were significantly reduced by 5-39% (P < 0.05 or less for all) by stanol esters; the higher their baseline proportions the higher were their reductions. The precursor sterol proportions were significantly increased by 10- 46%, and their high baseline levels predicted low reduction of serum cholesterol. The decrease of the scheduled stanol dose from 3 to 2 g/day after 6-month feeding increased serum cholesterol by 5% (P < 0. 001) and serum plant sterol proportions by 8-13% (P < 0.001), but had no consistent effect on precursor sterols. In twelve subjects, the 12-month level of LDL cholesterol exceeded that of baseline; the non-cholesterol sterol proportions suggested that stimulated synthesis with relatively weak absorption inhibition contributed to the non-responsiveness of these subjects. In conclusion, plant stanol ester feeding lowers serum cholesterol in about 88% of subjects, decreases the non-cholesterol sterols that reflect cholesterol absorption, increases the sterols that reflect cholesterol synthesis, but also slightly increases serum plant stanols. Low synthesis and high absorption efficiency of cholesterol results in the greatest benefit from stanol ester consumption. (+info)
Oxidation of medium-chain acyl-CoA esters by extracts of Aspergillus niger: enzymology and characterization of intermediates by HPLC.
(8/2031)
The activities of beta-oxidation enzymes were measured in extracts of glucose- and triolein-grown cells of Aspergillus niger. Growth on triolein stimulated increased enzyme activity, especially for acyl-CoA dehydrogenase. No acyl-CoA oxidase activity was detected. HPLC analysis after incubation of triolein-grown cell extracts with decanoyl-CoA showed that beta-oxidation was limited to one cycle. Octanoyl-CoA accumulated as the decanoyl-CoA was oxidized. Beta-oxidation enzymes in isolated mitochondrial fractions were also studied. The results are discussed in the context of methyl ketone production by fungi. (+info)