The effect of 6 months supplementation with conjugated linoleic acid on insulin resistance in overweight and obese.

BACKGROUND: Contradicting results have been published regarding the effect of conjugated linoleic acid (CLA) on insulin resistance. However, only a few studies have used the euglycemic hyperinsulinemic clamp method, which is considered the standard for measuring insulin resistance. OBJECTIVE: To evaluate if CLA as a mixture of the main isomers trans-10 cis-12 and cis-9 trans-11 affects the insulin resistance in healthy overweight and obese male and female adults. DESIGN: The main study was a randomized, double-blind, placebo-controlled trial with change in body composition as primary end point comprising 118 subjects receiving supplementation with either placebo (olive oil) or CLA (Clarinol) for 6 months. A sub-population of 49 subjects agreed additionally to participate in an euglycemic hyperinsulinemic clamp study at baseline and after 6 months of supplementation with study drug. The primary outcome was the change in glucose uptake (M) as measured by the hyperinsulinemic euglycemic glucose clamp method. Secondary outcomes were the correlates between insulin resistance and changes in body composition or blood chemistry parameters. Forty-one subjects completed the clamp test at both time points. RESULTS: The median M of the CLA group was 11.0 mg min(-1) lean body mass (lbm)(-1) (n=24) at baseline, 10.3 mg min(-1) lbm(-1) (n=24) after 6 months, and the median difference was +0.21 mg min(-1) lbm(-1) (n=24). The median M of placebo group was 8.4 mg min(-1) lbm(-1) at baseline and 9.3 mg min(-1) lbm(-1) after 6 months and the median difference was -0.22 mg min(-1) lbm(-1) (n=17). No significant (P<0.05) differences were found within groups or between groups. Likewise, the glucose uptake insulin concentration ratio during clamp (M/I) was independent of treatment and time. Homeostasis model assessment (HOMA) and quantitative insulin sensitivity check index derived from fasting glucose and insulin were also independent of treatment and time, and HOMA for the clamp population (n=49) corresponded well with HOMA for the per protocol population (n=83). Correlation analysis showed that changes in M were inversely correlated to changes in glucohemoglobin (P=0.002), but did not correlate with changes in either glucose, insulin, insulin c-peptide, leptin, adiponectin or percent body fat. CONCLUSIONS: CLA does not affect glucose metabolism or insulin sensitivity in a population of overweight or obese volunteers.

Efficacy of injection vaccines against Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum).

Efficacy of mineral oil-based experimental injection vaccines against Flavobacterium psychrophilum were tested in rainbow trout, Oncorhynchus mykiss (Walbaum), under laboratory and field conditions. The vaccines consisted of formalin- or heat-inactivated whole bacterium cell preparations of two different serotypes (Fd and Th) or a combination of serologically different F. psychrophilum (Fd and/or Th and/or Fp(T);Th). Specific antibody responses against the bacterium in plasma and skin mucus were evaluated post-vaccination with enzyme-linked immunosorbent assay. Efficacy of the vaccinations was determined by challenge trials to F. psychrophilum with the vaccinated rainbow trout. Significantly higher antibody levels in plasma were detected in vaccinated fish compared with mock-vaccinated fish. Injection vaccination did not trigger specific antibody production in the skin mucus. Significantly higher survival of i.p. vaccinated fish compared with non-vaccinated fish was observed during the challenge. The results suggest that mineral oil-based injectable vaccines containing formalin- or heat-inactivated virulent cells of F. psychrophilum effectively triggered specific antibody production and protected the fish against bacterial cold water disease.

Relevance of incubation temperature for Vibrio salmonicida vaccine production.

AIMS: To investigate the relationships between water temperature, bacterial growth, virulence and antigen expression in Vibrio salmonicida, the causal agent of cold water vibriosis in Atlantic salmon (Salmo salar L.). METHODS AND RESULTS: The significance of sea temperature was investigated using historical clinical and oceanographic data. An upper threshold for disease of approx. 10 degrees C was established. The effects of culture temperature and media type on bacterial growth were studied on solid and in liquid media. The highest rates of cell division were identified at 15 degrees C on solid media and 10 degrees C in liquid media. Outer membrane protein (OMP) expression and serological response in Atlantic salmon were studied using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. A novel 76-kDa OMP produced in unshaken cultures at 10 degrees C was not found to stimulate a specific humoral response. CONCLUSIONS: Diagnostic agar plate-based incubation of suspected V. salmonicida should be carried out at 15 degrees C. High yield broth cultures for vaccine production should be incubated at 10 degrees C or lower. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is, to the best of our knowledge, the first to identify different optimal temperatures in a bacterial species cultured on physically different types of media. The evidence presented suggests that V. salmonicida and possibly other bacteria destined for vaccine use in poikilothermic organisms should be cultured at temperatures consistent with that at which disease occurs.

L-[3H]glutamate binding to a membrane preparation from crayfish muscle.

The binding of L-[3H]glutamate to an isolated membrane preparation from crayfish tail muscle has been studied. The muscle homogenate was osmotically shocked, frozen and thawed, and thoroughly washed before incubation with L-[3H]glutamate. The preparation showed high specific binding of L-glutamate with a KD of 0.12 microM and Bmax of 4.7 pmol/mg protein measured in Tris/HCl pH 7.3 and at 4 degrees C. Nonspecific binding was 5-10% of total binding. The glutamate binding was highly stereospecific [K0.5 (D-glutamate), 270 microM] and showed a high degree of discrimination between L-glutamate and L-aspartate [K0.5 (L-aspartate), 54 microM]. In mammalian CNS preparations potent agonists of L-glutamate such as kainate and N-methyl-D-aspartate had no effect at 1 mM, and quisqualate was a weak inhibitor of L-glutamate binding [K0.5 (quisqualate), 162 microM]. Ibotenate was the most potent inhibitor [K0.5 (ibotenate), 0.27 microM], and various esters of L-glutamate were of intermediate potency as displacers of L-[3H]glutamate binding (K0.5 values from 6 to 60 microM). The glutamate binding site from crayfish muscle is clearly different from any of the subclasses of glutamate receptors in mammalian CNS. A possible physiological function of the binding site is a postsynaptic receptor for glutamate, either an extra-junctional or a junctional receptor.

Fluorescence studies on dopamine beta-monooxygenase: effects of salts, pH changes, metal-chelating agents and Cu2+.

The intrinsic protein fluorescence of dopamine beta-monooxygenase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) has been characterized. The fluorescence is dominated by emission from tryptophans in a hydrophobic environment. Changes in the conformation of the enzyme induced by anions, pH changes, metal-chelating agents and Cu2+ have been determined. Conformational transitions induced by anions take place at concentrations between 0.05 and 0.2 M. Most anions give rise to a blue-shift, while ClO4- induces a red-shift of the emission spectrum. pH dependence of the protein fluorescence revealed a conformational change between pH 6.0 and 5.0. The interactions between dopamine beta-monooxygenase and seven different metal-chelating agents have been investigated using protein fluorescence, heat inactivation, and inhibition measurements. All the metal-chelating agents are able to remove the active-site copper as demonstrated by complete inhibition of enzyme activity, restoration of activity by the addition of copper, and the observation that the enzyme becomes more sensitive to heat inactivation in the presence of chelating agents, thus behaving similarly to the copper-free apoenzyme. The charge and size of the chelating agents are of importance for the reaction with the active-site copper, which is consistent with a mechanism for removal of the copper, including a ternary enzyme-copper chelating agent complex. By contrast, under turnover conditions in the presence of substrates, dissociation of the active-site copper and chelation of the free copper is a dominating mechanism. Three distinct conformations were characterized on the basis of the fluorescence spectra and the degree of quenching by Cu2+ and I-. For the copper-free apoenzyme a unique binding site for binding of the first copper was demonstrated by larger quenching of the protein fluorescence than for binding of additional copper.

Studies on the binding of copper to dopamine beta-monooxygenase and other proteins using the Cu2+ ion-selective electrode.

The binding of Cu2+ to native and copper-free dopamine beta-monooxygenase has been investigated by potentiometric titrations using a Cu2+-selective electrode. Stoichiometric formation constants have been determined from regression analysis of the resulting titration curves. The results establish a stoichiometry of four high-affinity binding sites for Cu/+ (log Kf approximately 11) per enzyme tetramer, and more binding sites of lower affinity (log Kf approximately 5-7). The data for binding of the first four Cu2+ to the enzyme tetramer indicate interactions in the binding to the sites. Bovine serum albumin, metal-free carbonic anhydrase, and ovotransferrin have also been titrated with Cu2+, and the formation constants of both high-affinity binding sites and other sites have been determined. The stoichiometry of one high-affinity binding site of Cu2+ for carbonic anhydrase (log Kf approximately 10-12) and two sites for ovotransferrin (log Kf approximately 11) agree with the reported metal binding properties of these proteins. The number of high-affinity binding sites for bovine serum albumin was pH dependent.

2,2-dipyridyl binding to metal substituted horse liver alcohol dehydrogenase.

The binding of 2,2-dipyridyl to metal substituted horse liver alcohol dehydrogenase was measured by spectrophotometric titrations. Large changes in the visible absorption spectra were seen for the Co2+, Cu2+ and Ni2+ hybrids upon coordination of 2,2-dipyridyl, due to a change in coordination number. The formation constants for binding to the Co2+ and Cd2+ hybrids are of the order 10(6) M-1, which means that these hybrids have a 500-fold higher affinity for 2,2-dipyridyl than the native Zn2+ enzyme. 2,2-dipyridyl has a 100-fold higher affinity for enzyme bound Cd2+ than for aqueous Cd2+ ions, while for Cu2+ and Zn2+ the opposite is the case. None of the substituted metal ions were removed from the active site during titration with the chelator 2,2-dipyridyl.

The binding of sulfonamides to horse liver alcohol dehydrogenase.

The binding of sulfonamides to the active site of horse liver alcohol dehydrogenase has been studied by their effect on affinity labelling and steady state kinetics. Affinity labelling with iodoacetate and BIP has been used to study binding to free enzyme. The unsubstituted sulfonamide, sulfanilamide (I), shows very weak binding compared to the other sulfonamides tested. Most important for binding is the type of substituent attached to the parent sulfonamide, particularly when as in sulfathiazole this is a heterocycle which binds to the catalytic zinc atom of the enzyme. For sulfathiazole the dissociation constant from the enzyme is pH dependent showing two pKa values. The lower at pH 7 is the pKa of the drug itself, while that at pH 9 agrees with the ionization of water bound to the catalytic zinc ion. Steady state kinetics have been carried out at pH 7.0 and 10.0 to examine sulfonamide binding to the enzyme when coenzyme is attached. Both NAD+ and NADH induce substrate competitive sulfonamide binding. Likewise sulfathiazole accelerates the dissociation of NADH from the enzyme and SO Vmax for alcohol oxidation. The latter like stimulation of the affinity labelling reaction with iodoacetate is considered to result from binding of the thiazole ring to the catalytic zinc ion. With all the sulfonamides examined hydrophobic binding and charge are important in determining affinity to the active site and the mode of binding. Sulfonamides containing pyrazole or imidazole rings can be important in alcohol therapy.

Binding of ligands to the catalytic zinc ion in horse liver alcohol dehydrogenase.

The affinity of nitrogen and sulfur ligands for the catalytic zinc ion in horse liver alcohol dehydrogenase has been investigated by their influence on the affinity labeling reaction with iodoacetate. All the nitrogen compounds including ammonia, a primary and a secondary amine, and heterocycles containing a pyridine-type nitrogen with the exception of 2,2-dipyridyl were found to activate the affinity labeling reaction. Activation results from inner-sphere ligand coordination to the catalytic zinc ion. Closely related pyridine compounds gave a regular increase in affinity for the enzyme with increasing basicity, as expected for coordination to a metal ion. The sulfur compounds penicillamine and mercaptoethanol also activated the affinity labeling reaction, but dimercaptopropanol bound very tightly as a bidentate inhibited the reaction. The anions hydrosulfide, diethyldithiocarbamate, and cyanide coordinated to the catalytic zinc ion, whereas azide, thiocyanate, tetrazole, and iodide complexed the anion-binding site. The anionic metal ligands increased the rate of inactivation of the enzyme with iodoacetamide by binding to the catalytic zinc ion, while the binding of iodoacetate to the anion-binding site was prevented.

Ionization-linked cooperative interactions in the active site of horse liver alcohol dehydrogenase.

Affinity labeling of horse liver alcohol dehydrogenase with iodoacetate in the presence of the activator imidazole has been studied from pH 6.1 to 10.5. The pH profiles for the dissociation constants of iodoacetate from the free enzyme and the enzyme-imidazole complex and of imidazole from the free enzyme and the binary enzyme-iodoacetate complex were determined. The variation with pH of the dissociation constants of iodoacetate (KI) and imidazole (KL) have in common a pKa of 8.6 assigned to the zinc-water ionization, and a pKa near 10. Lysine modification by ethyl acetimidate results in a higher affinity of iodoacetate to the enzyme at high pH as the pKa values of the lysine residues are increased. The binding of iodoacetate and imidazole at each enzyme subunit shows negative cooperativity at pH less than 9, with an interaction constant of 4.8 at pH 6.1. Positive cooperativity is observed at pH greater than 9, with an interaction constant of 0.5 at pH 10.5. The pH-dependent change in cooperativity results from the removal of the zinc-water ionization when imidazole becomes coordinated to the catalytic zinc ion. When iodoacetate binds at the anion binding site, a large perturbation of the zinc-water ionization is observed. Unlike imidazole, the binding of 1,10-orthophenanthroline and iodoacetate shows positive cooperativity at both pH 8.2 and 10.0 with an interaction constant as low as 0.06 at pH 10.0.

Oxidation of two hydroxylated ochratoxin A metabolites by alcohol dehydrogenase.

(4R)-4-hydroxyochratoxin A, (4S)-4-hydroxyochratoxin A, and 10-hydroxyochratoxin A, all formed from ochratoxin A, were incubated with alcohol dehydrogenase in the presence of NAD. Only (4R)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A acted as substrates for the enzyme. K(m) and turnover number for 10-hydroxyochratoxin A were 110 muM and 0.1 s(-1), respectively.

Affinity labelling of liver alcohol dehydrogenase. Effects of pH and buffers on affinity labelling with iodoacetic acid and (R, S)-2-bromo-3-(5-imidazolyl)propionic acid.

Both iodoacetic acid and (R,S)-2-bromo-3-(5-imidazolyl)propionic acid (BrImPpOH) react with liver alcohol dehydrogenase in an affinity labelling mechanism between pH 6.1 and 10.5. The buffer-independent dissociation constants and the first-order rate constants have been determined as a function of pH. With BrImPpOH a pKa close to 9 for the free enzyme is assigned to the zinc-water ionization. The buffers used exerted a protective effect upon the inactivation of the enzyme by iodoacetic acid and BrImPpOH. Phosphate buffer showed a high degree of protection especially at lower pH, while zwitterionic buffers like Mes (4-morpholineethanesulfonic acid), Pipes (1,4-piperazinediethanesulfonic acid), Epps [4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid] and Bicine [N,N-bis(2-hydroxyethyl)glycine] gave less protection to various degrees. An exception was Ches (cyclohexylaminoethanesulfonic acid) which had an anomalously high affinity for the iodoacetate binding site. The dissociation constants of the buffers were calculated for the case of inactivation by both iodoacetic acid and BrImPpOH.