A B S T R A C T Porcine intestinal mucosal heparin induced aggregation of platelets in citrated platelet-rich plasma and enhanced platelet aggregation and serotonin secretion induced by other agents. This action ofheparin was blocked by substances that elevate platelet cyclic AMP and by EDTA but not by inhibitors of platelet cyclooxygenase. The effect was not inhibited by apyrase or by N-amylthio-5'-AMP and therefore did not require the action of ADP, nor was there activation of platelet phospholipase. Platelet aggregation by heparin required a plasma cofactor different from the cofactor required for ristocetin.Fractionation of heparin yielded preparations that varied in molecular weight and, within a given molecular weight fraction, in affinity for antithrombin III.Fractions of high molecular weight (average 20,000) were more reactive with platelets than were fractions of low molecular weight (7,000). Anticoagulant activity did not parallel the platelet reactivity of heparin fractions. Among high molecular weight fractions, preparations of high or low antithrombin affinity were equally active in induction of platelet aggregation. In low molecular weight fractions, there was an inverse relationship between platelet reactivity and anticoagulant activity in normal platelet-rich plasma, but, in plateletrich plasma depleted of antithrombin, low molecular weight fractions of high and low antithrombin affinity reacted equally with platelets. These results suggest that formation of an antithrombin-heparin complex protected platelets from aggregation by heparin.Selection of heparin fractions of low molecular weight and high antithrombin affinity may improve anticoagulant therapy and development of thromboresistant heparin-coated artificial materials.

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The antioxidant response element (ARE) found in the 5-flanking region of the rat quinone reductase gene has been further characterized by mutational and deletion analysis. The results indicate that the 31-base pair ARE, which contains a 13-base pair palindromic sequence, can be further separated into three regions, all three of which are required for elevated basal level gene expression. These three regions include the proximal and distal half-sites as well as a 3-flanking region consisting of 4 adenine nucleotides. Neither the proximal nor the distal half-site alone mediates transcriptional activation by ␤-naphthoflavone. However, when placed together the two half-sites restore responsiveness to the inducer. Interestingly, the presence of only 1 of the 4 adenine nucleotides in the 3-flanking region of the proximal halfsite is required for responsiveness to the inducer. Point mutations within the ARE indicate that several nucleotides in both the proximal and distal half-sites are required for basal level gene expression. Electrophoretic mobility shift analysis using the ARE as the probe indicates that enhancers found in the glutathione S-transferase Ya and P genes recognize a similar trans-acting factor(s) found in crude nuclear extracts from human Hep G2 cells. Further, this complex can be detected in nuclear extracts from rat liver and rat hepatoma cells but not in mouse Hepa 1c1c7 cells or in human HeLa cells. The ARE-nucleoprotein complex can also be detected in F9 cells which lack significant levels of Jun/Fos proteins. Although the rat ARE resembles the human quinone reductase ARE which contains a consensus TRE, the 2-nucleotide change in the core sequence (TGACTCA versus TGACTTG) eliminates the high affinity TRE motif in the rat ARE. The rat ARE forms a nucleoprotein complex in Hep G2 and other cells with different properties than AP-1. NAD(P)H:quinone oxidoreductase (DT-diaphorase, quinone reductase) is a phase II drug-metabolizing enzyme found in liver and other tissues and is inducible by a wide variety of compounds including phenolic antioxidants, planar aromatic compounds and TCDD 1 (1-3). In order to further understand the molecular mechanisms underlying the constitutive and inducible expression of this gene, two response elements found in the 5Ј-flanking region of the rat gene have been identified (4). One of these elements is the xenobiotic response element (XRE) found in the CYP1A1 and rat glutathione S-transferase Ya subunit genes (5, 6). The second element is the antioxidant response element (ARE) which is responsible for basal and inducible expression of the gene (4). We have demonstrated that diverse xenobiotics such as ␤-naphthoflavone (␤-NF), tert-butylhydroquinone (t-BHQ), hydrogen peroxide, and 12-O-tetradecanoylphorbol 13-acetate (TPA) can transcriptionally activate gene expression through the ARE (4, 7). A high affinity DNA-protein binding complex exists in crude nuclear extracts of Hep G2 cells based on DNase I footprinting, methylation interference and protection assays, and electroph...

A new simple and rapid method for quantitative semimicro determination of blood urea is presented. Diacetyl monoxime and thiosemicarbazide form in acid medium with urea a red color complex with a maximum adsorption at 530 mµ stable at room temperature and requiring 0.2 ml. of 1:10 blood filtrate.

Inhibition of BACE1 to prevent brain Aβ peptide formation is a potential disease-modifying approach to the treatment of Alzheimer’s disease. Despite over a decade of drug discovery efforts, the identification of brain-penetrant BACE1 inhibitors that substantially lower CNS Aβ levels following systemic administration remains challenging. In this report we describe structure-based optimization of a series of brain-penetrant BACE1 inhibitors derived from an iminopyrimidinone scaffold. Application of structure-based design in tandem with control of physicochemical properties culminated in the discovery of compound 16, which potently reduced cortex and CSF Aβ40 levels when administered orally to rats.