There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood-brain barrier (BBB) using well-established in vitro models (brain endothelial cell lines and ECV304 monolayers co-cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevant in vivo metabolites, as well as the dietary anthocyanins and in vivo forms, cyanidin-3-rutinoside and pelargonidin-3-glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (P app ) of the citrus flavonoids, hesperetin and naringenin, across the in vitro BBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and its in vivo metabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity. Keywords: blood-brain barrier, flavonoids, glucuronidation, hesperetin, naringenin, neuroprotection. There is growing interest in dietary therapeutic strategies to combat oxidative stress-induced damage to the CNS associated with a number of pathophysiological processes, including Alzheimer's disease, cerebrovascular disease such as strokes or lesions, Parkinson's disease, Creutzfeldt-Jakob disease and certain traumas (Coyle and Puttfarcken 1993;Cantuti-Castelvetri et al. 2000). In addition, changes in the optimal performance of the CNS may occur simply as a function of ageing, possibly exacerbating the motor and cognitive behavioural changes seen in these conditions (Shukitt-Hale 1999). Recent studies have highlighted an important role for the neuroprotective actions of dietary components, including flavonoids found in fruit, vegetables and plant-derived beverages (for a review see Abbreviations used: BBB, blood-brain barrier; b.END5, brain endothelial cell line from mouse; bFGF, basic fibroblast growth factor; BSA, bovine serum albumin; C3R, cyanidin-3-rutinoside; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum; FCS, fetal calf serum; HPLC, high-performance liquid chromatography; MTT, 2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; OATP, organic anion transport polypeptide; P3G, pelargonidin-3-glucoside; P app , apparent permeability; PBS, phosphate-buffered saline; P-gp, P-glycoprotein; RBE4, brain endothelial cell line from rat; RT, retention time; TEER, transendothelial electrical resistance.

Purpose: Angiogenesis and vascular endothelial growth factor (VEGF) expression are associated with a poor outcome in bladder cancer.To understand more about the mechanisms, we studied the role of delta-like 4 (DLL4), an endothelial-specific ligand of the Notch signaling pathway, in bladder cancer angiogenesis. Experimental Design: The expression of DLL4, CD34, and VEGF were studied in a cohort of 60 bladder tumors and 10 normal samples using quantitative PCR. In situ hybridization was used to study the pattern of DLL4 expression in 22 tumor and 9 normal samples. Serial sections were also stained for CD34 and a-smooth muscle actin (a-SMA) using conventional immunohistochemistry.Results: The expression of DLL4 was significantly up-regulated in superficial (P < 0.01) and invasive (P < 0.05) bladder cancers. DLL4 expression significantly correlated with CD34 (P < 0.001) and VEGF (P < 0.001) expression. The in situ hybridization studies showed that DLL4 was highly expressed within bladder tumor vasculature. Additionally, DLL4 expression significantly correlated with vessel maturation as judged by periendothelial cell expression of a-SMA, 98.7% of DLL4-positive tumor vessels coexpressed a-SMA, compared with 64.5% of DLL4-negative tumor vessels (P < 0.001). High DLL4 expression may have prognostic value in superficial and invasive bladder. Conclusion: DLL4 expression is associated with vascular differentiation in bladder cancer; thus, targeting DLL4 may be a novel antiangiogenic therapy.

Abstract. The pathogenesis of cerebral malaria is poorly understood. One hypothesis is that activation of microglia and astrocytes in the brain might cause the cerebral symptoms by excitotoxic mechanisms. Cerebrospinal fluid was sampled in 97 Kenyan children with cerebral malaria, 85% within 48 hr of admission. When compared with an agematched reference range, there were large increases in concentrations of the excitotoxin quinolinic acid (geometric mean ratio cerebral malaria/reference population [95% confidence limits] ϭ 14.1 [9.8-20.4], P Ͻ 0.001) and total neopterin (10.9 [9.1-13.0], P Ͻ 0.001) and lesser increases in tetra-hydrobiopterin, di-hydrobiopterin, and 5-hydroxyindoleacetic acid. There was no change in tryptophan concentration. In contrast, nitrate plus nitrite concentrations were decreased (geometric mean ratio ϭ 0.45 [0.35-0.59], P Ͻ 0.001). There was a graded increment in quinolinic acid concentration across outcome groups of increasing severity. The increased concentration of quinolinic acid suggests that excitotoxic mechanisms may contribute to the pathogenesis of cerebral malaria.Cerebral malaria is the most serious complication of infection with Plasmodium falciparum, which causes the death of more than 1 million children in sub-Saharan Africa each year. 1 The pathogenesis of childhood cerebral malaria is not well understood. 2 A central occurrence in the production of the clinical syndrome is adherence of parasitized erythrocytes to endothelial cells of the cerebral vasculature. This initiates a cascade of events that cause intense endothelial activation on the luminal side of the blood-brain barrier. 3 These events include cytoadherence itself, 4 and the production of inflammatory cytokines, 5-7 malarial toxins, 8 and nitric oxide.9-12 Much less is known about events occurring on the abluminal side of the blood-brain barrier in cerebral malaria and the cause of cerebral symptoms.The clinical syndrome of childhood cerebral malaria is characterized by the rapid onset and recovery from a diffuse encephalopathy. 13 The coma is complicated by raised intracranial pressure and seizures occur in more than half the patients. 14,15 Between 5% and 15% of survivors of cerebral malaria have neurologic sequelae suggesting focal brain damage. 16,17 These clinical findings could be explained by an excitotoxic mechanism.Experimental stimulation of cells of the macrophage/ monocyte lineage by various cytokines causes the parallel induction of indoleamine-pyrrole 2,3-dioxygenase (EC 1.13.11.42, indoleamine 2,3-dioxygenase), GTP cyclohydrolase I (EC 3.5.4.16), and nitric oxide synthase (EC 1.14.13.39). 18,19 These enzymes catalyze the first step of pathways that lead to the formation of quinolinic acid, neopterin and tetrahydrobiopterin, and nitric oxide, respectively. In the brain, microglia constitute the resident macrophage/ monocyte cells. Experimental stimulation of microglia with cytokines also causes induction of these enzymes. 20,21 Quinolinic acid is an endogenous excitotoxin that is a selective agon...

Mitochondrial cytochrome oxidase is competitively and reversibly inhibited by inhibitors that bind to ferrous heme, such as carbon monoxide and nitric oxide. In the case of nitric oxide, nanomolar levels inhibit cytochrome oxidase by competing with oxygen at the enzyme's heme-copper active site. This raises the K(m) for cellular respiration into the physiological range. This effect is readily reversible and may be a physiological control mechanism. Here we show that a number of in vitro and in vivo conditions result in an irreversible increase in the oxygen K(m). These include: treatment of the purified enzyme with peroxynitrite or high (microM) levels of nitric oxide; treatment of the endothelial-derived cell line, b.End5, with NO; activation of astrocytes by cytokines; reperfusion injury in the gerbil brain. Studies of cell respiration that fail to vary the oxygen concentration systematically are therefore likely to significantly underestimate the degree of irreversible damage to cytochrome oxidase.

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Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome.