We have analyzed the modulation of amyloid (3-protein precursor (APP) gene expression in human umbilical vein endothelial cells (HUVEC). The level of the APP mRNA transcripts increased as HUVEC reached confluency. In confluent culture the half-life of the APP mRNA was 4 hr. Treatment of the cells with human recombinant interleukin 1 (IL-1), phorbol 12-myristate 13-acetate, or heparin-binding growth factor 1 enhanced the expression of APP gene in these cells, but calcium ionophore A23187 and dexamethasone did not. The protein kinase C inhibitor 1-(isoquinolinsulfonyl)-2-methylpiperazine (H7) inhibited IL-1-mediated increase of the level of APP transcripts. To map IL-1-responsive elements of the APP promoter, truncated portions of the APP promoter were fused to the human growth hormone reporter gene. The recombinant plasmids were transfected into mouse neuroblastoma cells, and the cell medium was assayed for the human growth hormone. A 180-base-pair region of the APP promoter located between position -485 and -305 upstream from the transcription start site was necessary for IL-1-mediated induction of the reporter gene. This region contains the upstream transcription factor AP-1 binding site. These results suggest that IL-1 upregulates APP gene expression in HUVEC through a pathway mediated by protein kinase C, utilizing the upstream AP-1 binding site of the APP promoter.The amyloid A3-protein precursor (APP) gene codes for a family of proteins, each containing a 42-amino acid fragment called amyloid 3 or A4 protein, that is found in brains and the cerebrovasculature of Alzheimer disease, Down syndrome, hereditary cerebral hemorrhage (Dutch type), and aging (1)(2)(3)(4)(5). The APP gene is transcriptionally active in a variety of tissues, highly conserved in evolution, maps to human chromosome 21 (6-10), and probably has no mutations associated with Alzheimer disease (11, 12). Transcripts containing three similar but nonidentical open reading frames have been identified (13)(14)(15)(16)(17). In situ hybridization studies show a very complex pattern of the APP gene expression in brain (18-23).Different levels of APP mRNAs were detected in neurons, some glial cells, and cells that compose blood vessels, presumably some endothelial cells (23). The detection of APP mRNA in these cells strengthens the possibility that at least one form of amyloid 83-protein-cerebrovascular-could originate locally from blood vessels (24, 25).The importance ofunderstanding the regulation ofthe APP gene is underscored by two observations. First, there are differences between Alzheimer disease patients and controls in the levels of APP mRNAs in certain brain regions (19,23,(26)(27)(28)(29)(30). Second, the difference in the level of APP mRNAs between trisomy 21 Down-syndrome patients and controls having two chromosomes 21 was higher than the expected 3:2 ratio (9). In the absence of structural mutations, aberrant levels of the precursor protein may contribute to pathology. These observations suggest that there is aberrant regulation...

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New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses ~-[carboxy-'~C]tyrosine as the substrate. I4CO2 is released from the products of the hydroxylation and further metabolism of ~-[carboxy-'~C]tyrosine by incubation with ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.It is well established that tyrosinase from lower organisms catalyses the first two steps of melanin synthesis: the hydroxylation of tyrosine to dopa, and the oxidation of dopa to dopaquinone [l]. The classical view is that this is also true for the mammalian enzyme (21; it has been suggested that mammalian tyrosinase may catalyse a third step in melanin synthesis: the oxidation of 5,6-dihydroxyindole [3, 41. There have, however, been reports of the separation of melanogenic tyrosine hydroxylase and dopa oxidase activities on purification of the mammalian enzyme [5, 61. To determine conclusively whether the tyrosine hydroxylase and dopa oxidase activities of tyrosinase are inherent in one protein molecule, satisfactory assays for these two activities are required. There are many problems associated with the existing assays so the aim of this work was to develop improved assays for both activities. Tyrosine hydroxylase assayThe tyrosine hydroxylase activity of tyrosinase requires Ldopa, the product of the reaction, as a cofactor. In the absence of cofactor, no activity [7] or very low activity [8] has been reported. In general, a cofactor is required at a low concentration relative to the substrate, but a relatively high concentration of L-dopa is required [9]. If tyrosinase does catalyse the first two steps of melanin synthesis, addition of L-dopa as a cofactor also represents addition of a competitive substrate. Oxidation of L-dopa by dopa oxidase activity will deplete the concentration of cofactor. The requirement for L-dopa as a cofactor thus makes the tyrosine hydroxylase activity of tyrosinase difficult to assay.The Pomerantz method [lo] is used most frequently to assay the tyrosine hydroxylase activity of tyrosinase. It uses ~-[3,5-~H]tyrosine as the substrate and measures the 3 H 2 0Correspondence to A.

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