DHA-enriched re-esterified triacylglycerol fish oil supplementation and oily fish consumption enhance red blood n-3 fatty acid index in Omani pre-adolescent schoolchildren.

Dietary habits of Omani population particularly of children and young adults have changed significantly. Consumption of imported calorie-dense foods, vegetable oils, milled and polished grains and carbonated beverages have become the norm. Concomitantly, there has been an exponential increase in the prevalence of non-communicable diseases. The impact of the westernisation of eating habits on children has not been evaluated. We have investigated blood fatty acid profile of male (n = 125) and female (n = 160) children aged 9 and 10 (9.8 ±â€¯0.4) years enrolled from three state-funded schools. The schools, which are homogenous with respect to socio-economic background of their pupils, were randomised into fish oil (n = 98), oily fish (n = 82) or control (n = 105) group. Subsequently, the children were given during morning tea break for 12 weeks: 1. DHA-enriched re-esterified triacylglycerol fish oil capsule with cheese/salad sandwich (fish oil group), 2. Lightly grilled oily fish with salad (fish group) or 3. Cheese/salad sandwich (control group). At baseline, the males had higher myristic, palmitic and oleic and lower adrenic acids than the females (p < 0.05). There was no difference in n-3 fatty acid index (4.86 ±â€¯1.95 vs. 5.12 ±â€¯1.67, p > 0.05) or AA (14.6 ±â€¯1.9 vs. 14.9 ±â€¯1.8, p > 0.05) between the genders. There was no difference in any of the fatty acids between the three groups at baseline. Post-intervention, the oily fish group had lower n-3 fatty acid index (EPA + DHA, 6.03 ±â€¯1.39 vs. 6.60 ±â€¯1.63, p < 0.05) and higher AA (15.2 ±â€¯1.8 vs. 13.7 ±â€¯2.0, p = 0.0001) and n-3 DPA (1.40 ±â€¯0.27 vs. 1.07 ±â€¯0.22, p = 0.0001) compared with those who received fish oil capsules. In both the fish oil and oily fish groups, fatty acid index correlated positively with AA (r = 0.394, p = 0.0001; r = 0.231, p = 0.038) and negatively with total saturated (r = - 0.816, p = 0.0001; r = - 0.439, p = 0.0001) and total mono-unsaturated (r = - 0.431, p = 0.0001; r = - 0.231, p = 0.037) fatty acids. Although seafood is an integral part of traditional Omani cuisine the children had a low level of n-3 fatty acids index. There is a need to address this nutritional insufficiency through school feeding programme, targeted intervention with n-3 fatty acid enriched food products and/or family education programme.

Mass fragmentation study of the trimethylsilyl derivatives of arctiin, matairesinoside, arctigenin, phylligenin, matairesinol, pinoresinol and methylarctigenin: their gas and liquid chromatographic analysis in plant extracts.

The mass fragmentation patterns and the characteristic behavior of the trimethylsilyl (TMS) derivatives of the dibenzylbutyrolactone-type (arctiin, arctigenin, methylarctigenin, matairesinoside, matairesinol) and those of the diphenylperhydrofurotetrahydrofurane-type (phylligenin, pinoresinol) lignans, obtained by gas chromatography-mass spectrometry (GC-MS), were presented. It was shown that upon acidic hydrolysis the dibenzylbutyrolactone-type lignans are stable while the diphenylperhydrofurotetrahydrofurane-type ones decompose. As a novelty to the field we confirmed that the fragment species of the derivatized lignan glycosides, in the presence of excess hexamethyldisilazane, leaded to their in situ derivatization. Quantification of the selective fragment ions of the TMS derivatives by GC-MS, in respect of the ions found one by one, and concerning the selective fragment ions {SFI(s)} in total, provided acceptable reproducibilities, suitable for quantitation purposes: varying between 1.20% and 6.6% relative standard deviation percentages (RSD%). For characterization of the behavior of various type of lignans, analyses were performed with the untreated and with the trifluoroacetic acid hydrolyzed plant extracts, from the same sample, in parallel, both by GC-MS and by high performance liquid chromatography-mass spectrometry, working in the positive electron ionization mode (HPLC-ESPI-MS). The analysis of lignans in fruit and leaf extracts (obtained from the Arctium, Centaurea and Forsythia plants) was confirmed both by GC-MS and by HPLC-ESPI-MS. Our multicomponent system (including the identification and quantification of sugars, sugar alcohols, and several members of various homologous series of acids, anthraquinones and flavonoids) has been extended to the analysis of lignan glycosides and to the free lignans. Reproducibilities in the quantitation of lignans in plant matrices, as averages on GC and HPLC basis, varied between 0.9% and 11% (RSD). The distribution of the lignan constituents was presented for 5 Arctium, for 8 Centaurea and for 4 Forsythia plant extracts: the total of lignan contents varied between 0.42 and 87.9 mg/g, respectively.

pH-induced changes in activity and conformation of NADH oxidase from Thermus thermophilus.

Thermus thermophilus NADH oxidase (NOX) activity exhibits a bell-shaped pH-dependency with the maximal rate at pH 5.2 and marked inhibition at lower pH. The first pH transition, from pH 7.2 to pH 5.2, results in more than a 2-fold activity increase with protonation of a group with pKa=6.1+/-0.1. The difference in fluorescence of the free and enzyme-bound flavin strongly indicates that the increase in enzyme activity in a pH-dependent manner is related to a protein-cofactor interaction. Only one amino acid residue, His75, has an intrinsic pKa approximately 6.0 and is localized in proximity (<10 A) to N5-N10 of the isoalloxazine ring and, therefore, is able to participate in such an interaction. Solvent acidification leads to the second pH transition from pH 5.2 to 2.0 that results in complete inhibition of the enzyme with protonation of a group with an apparent pKa=4.0+/-0.1. Inactivation of NOX activity at low pH is not caused by large conformational changes in the quaternary structure as judged by intrinsic viscosity and sedimentation velocity experiments. NOX exists as a dimer even as an apoprotein at acidic conditions. There is a strong coupling between the fluorescence of the enzyme-bound flavin and the intrinsic tryptophans, as demonstrated by energy transfer between Trp47 and the isoalloxazine ring of flavin adenine dinucleotide (FAD). The pH-induced changes in intrinsic tryptophan and FAD fluorescence indicate that inhibition of the FAD-binding enzyme at low pH is related to dissociation of the flavin cofactor, due to protonation of its adenine moiety.

Effect of polyanion on the acidic conformational transition of native and denatured ferricytochrome c. Circular dichroism study.

Interaction of polyanion poly(vinylsulfate) with oxidized cytochrome c (cyt c) significantly affects the protein main characteristics. One of them, pKa value of acidic transition, was shifted from an apparent pKa value 2.5 (typical for cyt c in low ionic strength solvent) to approximately 5.20 +/- 0.15 upon polyanion binding to the protein, pointing to a likely involvement of histidines 26 and/or 33 in the protein acidic transition in complex with the polyanion. The acidic transition followed at 6 different wavelengths all over circular dichroism spectrum, monitoring different parts of the protein structure, revealed basically two-state character process. Only ellipticity at 262 nm indicated a low-cooperative pH-induced conformational transition in heme region with an apparent pKa approximately 4.34 +/- 0.25 in accordance with absorbance change at 620 nm. Polyanion also interacts with chemically-denatured (in the presence of 9 mol/l urea) state of the protein as it follows from stabilization of protein residual structure at acidic pH and its effect on pKa value of acidic transition of chemically-denatured cyt c. Destabilization effect of polyanions on native and, on the other hand, stabilization influence on partially unfolded conformations of the protein are discussed with an implication for their chaperone-like properties in vivo and in vitro.

Effect of the central disulfide bond on the unfolding behavior of elongation factor Ts homodimer from Thermus thermophilus.

Functionally active elongation factor Ts (EF-Ts) from Thermus thermophilus forms a homodimer. The dimerization interface of EF-Ts is composed of two antiparallel beta-sheets that can be connected by an intermolecular disulfide bond. The stability of EF-Ts from T. thermophilus in the presence and absence of the intermolecular disulfide bond was studied by differential scanning calorimetry and circular dichroism. The ratio of the van't Hoff and calorimetric enthalpies, delta H(vH)/delta H(cal), indicates that EF-Ts undergoes thermal unfolding as a dimer independently of the presence or absence of the disulfide bond. This can be concluded from (1) the presence of residual secondary structure above the thermal transition temperature, (2) the absence of concentration dependence, which would be expected for dissociation of the dimer prior to unfolding of the monomers, and (3) a relatively low heat capacity change (delta Cp) upon unfolding. The retained dimeric structure of the thermally denatured state allowed for the determination of the effect of the intermolecular disulfide bond on the conformational stability of EF-Ts, which is deltadelta G(S-S,SH HS) = 10.5 kJ/mol per monomer at 72.5 degrees C. The possible physiological implications of the dimeric EF-Ts structure and of the intersubunit disulfide bond for the extreme conformational stability of proteins in thermophiles are discussed.

Effect of N-domain on the stability of elongation factor Ts from Thermus thermophilus.

Elongation factor Ts (EF-Ts) from Thermus thermophilus forms a stable, functionally active homodimer in solution. Its monomer is composed of two domains: amino-terminal domain containing 50 amino acid residues and a larger, 146 residues long, C-domain which participates in dimerization of EF-Ts. Effect of removal of the N-domain on the conformational stability of EF-Ts has been studied. For comparison, the stabilities of both the full-length EF-Ts and its C-domain were studied by differential scanning calorimetry, electronic absorption and fluorescence spectroscopies over a pH range from 4 to approximately 13. Thermal denaturation of EF-Ts and of C-domain, followed by circular dichroism at 222 nm, at pH 7.0, and the pH dependence of the fluorescence of the single tryptophan 30 residue indicate a conformational instability of the N-domain. While N-domain does not affect the stability of full-length EF-Ts at acidic pH, its removal leads to stabilization of the rest of the protein at basic pH. This is reflected by higher values of transition temperatures and calorimetric enthalpies of C-domain as compared to the full-length EF-Ts. High mobility of the N-domain in alkaline pH conditions decreased the thermal stability of covalently linked C-domain of EF-Ts. An increase in intramolecular interactions at acidic pH together with a decrease of conformational entropies of the thermally denatured proteins most likely diminishes this destabilization effect.

Phospholipase A(2) digestion of cardiolipin bound to bovine cytochrome c oxidase alters both activity and quaternary structure.

Phospholipase A(2) from Crotalus atrox hydrolyzes all of the phospholipids that are associated with purified, detergent-solubilized cytochrome c oxidase; less than 0.05 mol cardiolipin (CL)(1) remains bound per mol enzyme. Coincident with the hydrolysis of cardiolipin is a reversible decrease of 45-50% in the electron transport activity of the dodecylmaltoside-solubilized enzyme. Full activity is recoverable (90-98%) by addition of exogenous cardiolipin, but not by either phosphatidylcholine or phosphatidylethanolamine. Unexpectedly, cleavage of cardiolipin causes the dissociation of both subunits VIa and VIb from the enzyme. These are the two subunits that form the major protein-protein contacts between the two monomeric units within the dimeric complex. Although hydrolysis of CL by phospholipase A(2) and loss of these subunits is linked, the reverse process does not occur, i.e., removal of subunits VIa and VIb does not cause dissociation of the two functionally important, tightly bound cardiolipins. Nor does addition of exogenous cardiolipin result in reassociation of the two subunits with the remainder of the complex. We conclude that cardiolipin is not only essential for full electron transport activity, but also has an important structural role in stabilizing the association of subunits VIa and VIb within the remainder of the bovine heart enzyme.

Molten globule-like state of cytochrome c induced by polyanion poly(vinylsulfate) in slightly acidic pH.

The effect of polyanion, poly(vinylsulfate), used as a model of negatively charged surface, on ferric cytochrome c (ferricyt c) structure in acidic pH has been studied by absorbance spectroscopy, circular dichroism (CD), tryptophan (Trp) fluorescence and microcalorimetry. The polyanion induced only small changes in the native structure of the protein at neutral pH, but it profoundly shifted the acid induced high spin state of the heme in the active center of cyt c to a more neutral pH region. Cooperativity of the acidic transition of ferricyt c in the presence of the polyanion was disturbed, in comparison with uncomplexed protein, as followed from different apparent pK(a) values observed in a distinct regions of the ferricyt c electronic absorbance spectrum (4.55+/-0.08 in the 620 nm band region and 5.47+/-0.15 in the Soret region). The ferricyt c structure in the complex with the polyanion at acidic pH (below pH 5.0) has properties of a molten globule-like state. Its tertiary structure is strongly disturbed according to CD and microcalorimetry measurements; however, its secondary structure, from CD, is still native-like and ferricyt c is in a compact state as evidenced by quenched Trp fluorescence. These findings are discussed in the context of the molten globule state of proteins induced on a negatively charged membrane surface under physiological conditions.

Coulombic and noncoulombic effect of polyanions on cytochrome c structure.

The properties of the complexes of ferricytochrome c with two different polyanions--poly(vinylsulfate) and poly(4-styrene-sulfonate)--with a comparable charge density but with the different size of the uncharged part of their molecules have been studied by means of optical spectroscopy, differential scanning colorimetry, and gel chromatography. Ferriccytochrome c formed a complex with the former one through coulombic interactions and remained in a native-like state. The addition of the second polyanion to a solution of ferric cytochrome c at a low ionic strength, pH 7.0, resulted in profound conformational change in the hydrophobic core of protein (opening of the heme crevice with a perturbation of the methionine 80-heme iron bond and the hydrophobic core of the protein). These may be understood as an involvement of noncoulombic (hydrophobic, H-bonding) interactions of the uncharged part of the polyanion molecule. Conformational changes and the observed shift in acidic transition from low spin to high spin state of ferric cytochrome c detected in the presence of the polyanions may have biological implication in understanding the origin of conformational changes in proteins induced in the course of their interaction with membrane lipids and membrane proteins.

Dimers of Thermus thermophilus elongation factor Ts are required for its function as a nucleotide exchange factor of elongation factor Tu.

Elongation factor Ts (EF-Ts) promotes the formation of active GTP-bound elongation factor Tu (EF-Tu) by accelerating the dissociation of GDP from the EF-Tu x GDP complex. Thermus thermophilus EF-Ts forms a dimer in solution, which is stabilised by interaction of a three-stranded antiparallel beta-sheet from each of the two EF-Ts molecules. A disulfide bridge and several hydrophobic interactions are the main structural elements which stabilise the dimer [Jiang, Y., Nock, S., Nesper, M., Sprinzl, M. & Sigler, P. B. (1996) Biochemistry 35, 10269-10278]. Site-directed mutagenesis was used to study the dimer formation and the effect of dimerization on the nucleotide exchange activity. The presence of the covalent disulfide bridge between the Cys190 residues has no effect on the activity. However, this disulfide bridge is not a necessary condition for the activity of EF-Ts. The amino acid residues Leu73, Cys190 and Phe192 form a hydrophobic core on the dimerization interface. Their replacement by Asp, Ala and Asp, respectively, influences to different degrees the stability of EF-Ts, the ability of EF-Ts to form dimers, and the ability to interact with EF-Tu. EF-Ts variants which were unable to form dimers were also inactive in the nucleotide exchange on EF-Tu. CD spectroscopy confirmed that this loss of activity is not due to changes in EF-Ts secondary structure. By calorimetric measurements, it was demonstrated that the dimer formation considerably contributes to the thermostability of T. thermophilus EF-Ts. Dimerization of T. thermophilus EF-Ts is required to fulfil its physiological function in protein biosynthesis and probably represents a strategy of the translation system in this thermophile to withstand high temperatures. The biochemical data presented here are supported by the recently solved structure of the T. thermophilus EF-Tu x EF-Ts complex [Wang, Y., Jiang, Y., Meyering-Voss, M., Sprinzl, M. & Sigler, P. B. (1997) Nature Struct. Biol. 4, 650-656] in which each EF-Tu in the dyad symmetrical heterotetrameric complex interacts with two subunits of EF-Ts.

Interaction of ferricytochrome c with polyanion Nafion.

The properties of the complex of ferricyt c with fluorosulfonated polyanion Nafion (as a representative 'hydrophobic' polyanion) have been studied by means of optical spectroscopy and differential scanning calorimetry. The addition of the polyanion to a solution of ferricyt c (pH 7.4) resulted in an expansion of the protein molecule characterized by a profound decrease in enthalpy of the thermal transition of ferricyt c. The conformational change of ferricyt c upon addition of Nafion was shown by a perturbation of the Met-80-heme iron bond and an apparent increase in the distance of Trp-59 from the heme. The conformational change in the heme region was also observed by examining the CD spectra. The conformational state of ferricyt c in a complex with Nafion was similar to that designated as state II by Hildebrandt (Hildebrandt, P. (1990) Biochim. Biophys. Acta 1040, 175-186) in the complex with negatively charged heteropolytungstates-a six-coordinated low-spin state with a destabilized structure of the heme crevice. The decrease in enthalpy of the thermal transition of ferricyt c, the spectral changes in absorbance and the CD spectra, together with an increase in Trp fluorescence induced by Nafion addition observed at high ionic strength, all point to the involvement of the non-coulombic interaction.

Effect of polyanions on ferricytochrome c.

Effects of two polyanions (heparin, Nafion) on absorption and CD spectra in the Soret region, and on fluorescence of ferricytochrome c were investigated. The conformational state of ferricyt c in the complexes with studied polyanions is significantly different. Whereas heparin affects the structure in the heme region of ferricyt c only slightly (ferricyt c remains in a native-like state), the influence of Nafion is more expressive (the Met-80-heme iron bond and the deeply buried region around Trp-59 are perturbed). Shift of the pK of the transition from neutral low-spin state to acidic high-spin state to higher pH values was observed.