Blueberries are rich in flavonoids, which possess antioxidant and anti-inflammatory properties. High flavonoid intakes attenuate age-related cognitive decline, but data from human intervention studies are sparse. We investigated whether 12 weeks of blueberry concentrate supplementation improved brain perfusion, task-related activation, and cognitive function in healthy older adults. Participants were randomised to consume either 30 mL blueberry concentrate providing 387 mg anthocyanidins (5 female, 7 male; age 67.5 ± 3.0 y; body mass index, 25.9 ± 3.3 kg·m −2 ) or isoenergetic placebo (8 female, 6 male; age 69.0 ± 3.3 y; body mass index, 27.1 ± 4.0 kg·m −2 ). Pre-and postsupplementation, participants undertook a battery of cognitive function tests and a numerical Stroop test within a 1.5T magnetic resonance imaging scanner while functional magnetic resonance images were continuously acquired. Quantitative resting brain perfusion was determined using an arterial spin labelling technique, and blood biomarkers of inflammation and oxidative stress were measured. Significant increases in brain activity were observed in response to blueberry supplementation relative to the placebo group within Brodmann areas 4/6/10/ 21/40/44/45, precuneus, anterior cingulate, and insula/thalamus (p < 0.001) as well as significant improvements in grey matter perfusion in the parietal (5.0 ± 1.8 vs -2.9 ± 2.4%, p = 0.013) and occipital (8.0 ± 2.6 vs -0.7 ± 3.2%, p = 0.031) lobes. There was also evidence suggesting improvement in working memory (2-back test) after blueberry versus placebo supplementation (p = 0.05). Supplementation with an anthocyanin-rich blueberry concentrate improved brain perfusion and activation in brain areas associated with cognitive function in healthy older adults.Key words: polyphenols, cognitive function, cerebral perfusion, fMRI, blueberry, aging.Résumé : Les bleuets contiennent beaucoup de flavonoïdes lesquels possèdent des propriétés antioxydantes et anti-inflammatoires. Un apport élevé en flavonoïdes atténue le déclin cognitif associé à l'âge, mais il y a peu d'études menées chez des humains. Nous analysons si 12 semaines de supplémentation d'un concentré de bleuets améliorent la perfusion cérébrale, l'activation cérébrale associée à une tâche et la fonction cognitive chez des personnes âgées en bonne santé. On répartit aléatoirement les participants en deux groupes, l'un (5 femmes, 7 hommes; âge 67,5 ± 3,0 ans; l'indice de masse corporelle 25,9 ± 3,3 kg·m −2 ) consommant 30 mL de concentré de bleuets procurant 387 mg d'anthocyanidines et l'autre (8 femmes, 6 hommes; âge 69,0 ± 3,3 ans; l'indice de masse corporelle 27,1 ± 4,0 kg·m −2 ), un placebo isoénergétique. Avant et après la supplémentation, les participants se soumettent à une batterie de tests de la fonction cognitive et au test numérique de Stroop dans un appareil de magnétique fonctionnelle 1.5T pour l'obtention en continu de clichés au moyen de la technique d'imagerie par résonance magnétique fonctionnelle. On détermine le volume de pe...

The redox-active dithiol/disulfide C315-Xaa-Xaa-C318 center has been implicated in the regulation of the human mitochondrial branched chain aminotransferase isozyme (hBCATm) [Conway, M. E., Yennawar, N., Wallin, R., Poole, L. B., and Hutson, S. M. (2002) Biochemistry 41, 9070-9078]. To explore further the mechanistic details of this CXXC center, mutants of the Cys residues at positions 315 and 318 of hBCATm were individually and in combination converted to alanine or serine by site-directed mutagenesis (C315A, C315S, C318A, C318S, C315/318A, and C315/318S). The effects of these mutations on cofactor absorbance, secondary structures, steady-state kinetics, and sensitivity toward hydrogen peroxide (H(2)O(2)) treatment were examined. Neither the UV-visible spectroscopic studies nor the circular dichroism data showed any major perturbations in the structure of the mutants. Kinetic analyses of the CXXC mutant proteins indicated primarily a modest reduction in k(cat) with no significant change in K(m). The largest effect on the steady-state kinetics was observed with the C315 single mutants, in which substitution of the thiol group resulted in a reduced k(cat) (to 26-33% of that of wild-type hBCATm). Moreover, the C315 single mutants lost their sensitivity to oxidation by H(2)O(2). The kinetic parameters of the C318 mutants were largely unaffected by the substitutions, and as with wild-type hBCATm, reaction of the C318A mutant protein with H(2)O(2) resulted in the complete loss of activity. In the case of oxidized C318A, this loss was largely irreversible on incubation with dithiothreitol. Mass spectrometry and dimedone modification results revealed overoxidation of the thiol group at position 315 to sulfonic acid occurring via a sulfenic acid intermediate in the H(2)O(2)-treated C318A enzyme. Thus, C315 appears to be the sensor for redox regulation of BCAT activity, whereas C318 acts as the "resolving cysteine", allowing for reversible formation of a disulfide bond.

This study presents the first three-dimensional structures of human cytosolic branched-chain aminotransferase (hBCATc) isozyme complexed with the neuroactive drug gabapentin, the hBCATc Michaelis complex with the substrate analog, 4-methylvalerate, and the mitochondrial isozyme (hBCATm) complexed with gabapentin. The branched-chain aminotransferases (BCAT) reversibly catalyze transamination of the essential branched-chain amino acids (leucine, isoleucine, valine) to ␣-ketoglutarate to form the respective branched-chain ␣-keto acids and glutamate. The cytosolic isozyme is the predominant BCAT found in the nervous system, and only hBCATc is inhibited by gabapentin. Pre-steady state kinetics show that 1.3 mM gabapentin can completely inhibit the binding of leucine to reduced hBCATc, whereas 65.4 mM gabapentin is required to inhibit leucine binding to hBCATm. Structural analysis shows that the bulky gabapentin is enclosed in the activesite cavity by the shift of a flexible loop that enlarges the active-site cavity. The specificity of gabapentin for the cytosolic isozyme is ascribed at least in part to the location of the interdomain loop and the relative orientation between the small and large domain which is different from these relationships in the mitochondrial isozyme. Both isozymes contain a CXXC center and form a disulfide bond under oxidizing conditions. The structure of reduced hBCATc was obtained by soaking the oxidized hBCATc crystals with dithiothreitol. The close similarity in active-site structures between cytosolic enzyme complexes in the oxidized and reduced states is consistent with the small effect of oxidation on pre-steady state kinetics of the hBCATc first half-reaction. However, these kinetic data do not explain the inactivation of hBCATm by oxidation of the CXXC center. The structural data suggest that there is a larger effect of oxidation on the interdomain loop and residues surrounding the CXXC center in hBCATm than in hBCATc. Pyridoxal 5Ј-phosphate (PLP)3 -dependent branched-chain aminotransferases (BCAT) reversibly catalyze the transfer of the ␣-amino group of the hydrophobic branched-chain amino acids (leucine, isoleucine, valine) to ␣-ketoglutarate to form the respective branched-chain ␣-keto acids and glutamate (1, 2). BCAT enzymes are found in both bacteria and higher organisms (3, 4). Mammals have a mitochondrial (BCATm) and a cytosolic (BCATc) form of the enzyme (5, 6), whereas bacteria have a single BCAT enzyme (7). Human mitochondrial BCATm (hBCATm) is expressed ubiquitously in body tissues, whereas human cytosolic BCATc (hBCATc) is found primarily in the nervous system (5, 8 -10). In fact, hBCATc is the predominant isozyme in brain, accounting for 60 -70% of total brain BCAT activity (8,11, 12). In the rat nervous system BCATc is expressed in selected populations of glutamatergic and ␥-aminobutyric acid (GABAergic) neurons (13), and Hutson and co-workers (14 -16) have postulated the existence of a glial-neuron nitrogen shuttle for the BCATs and branched-chain amino acids involved i...

The branched chain aminotransferase enzymes (BCAT) serve as nitrogen donors for the production of 30% of de novo glutamate synthesis in rat brain. Despite the importance of this major metabolite and excitatory neurotransmitter, the distribution of BCAT proteins in the human brain (hBCAT) remains unreported. We have studied this and report, for the first time, that the mitochondrial isoform, hBCATm is largely confined to vascular endothelial cells, whereas the cytosolic hBCATc is restricted to neurons. The majority of hBCATc-labelled neurons were either GABA-ergic or glutamatergic showing both cell body and axonal staining indicating a role for hBCATc in both glutamate production and glutamate release during excitation. Strong staining in hormone secreting cells suggests a further role for the transaminases in hormone regulation potentially similar to that proposed for insulin secretion. Expression of hBCATm in the endothelial cells of the vasculature demonstrates for the first time that glutamate could be metabolized by aminotranferases in these cells. This has important implications given that the dysregulation of glutamate metabolism, leading to glutamate excitotoxicity, is an important contributor to the pathogenesis of several neurodegenerative conditions, where the role of hBCATm in metabolizing excess glutamate may factor more prominently.

BCATc Cytosolic BCAT BCATm Mitochondrial BCAT BCKA Branched chain a-keto acids BCKD Branched chain a-keto acid dehydrogenase Cho Choline Cr Creatinine Glu Glutamate Glx Glu?glutamine GDH Glutamate dehydrogenase MSUD Maple syrup urine disease NAA N-acetyl aspartate NMDAR N-methyl-D-aspartate receptor NFTs Neurofibrillary tangles PLP Pyridoxal phosphate PSEN1 Presenilin 1 1 H MRS Proton magnetic resonance spectroscopy mI Myoinositol TBI Traumatic brain injury