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Converging lines of evidence implicate the beta-amyloid peptide (Ab) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce Ab production by functionally inhibiting g-secretase, the activity responsible for the carboxy-terminal cleavage required for Ab production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon Ab production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-di¯uoro-phenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP V717F reduces brain levels of Ab in a dose-dependent manner within 3 h. These studies represent the ®rst demonstration of a reduction of brain Ab in vivo. Development of such novel functional g-secretase inhibitors will enable a clinical examination of the Ab hypothesis that Ab peptide drives the neuropathology observed in Alzheimer's disease.

Here we show that this kinase is identical with or closely related to p38 (the mammalian homolog of HOG1 from yeast), a recently discovered protein kinase typically activated by inflammatory cytokines and environmental stress. Further, we demonstrate that activation of this kinase by thrombin is transient (with maximal stimulation at 1 min), is accompanied by tyrosine phosphorylation, and precedes the activation of the ERK kinases. This is the first report to show that p38 kinase is activated by thrombin and to suggest a role for this MAP kinase in the thrombin-mediated signaling events during platelet activation.We have recently shown that thrombin stimulates the activity of the MAP 1 kinases ERK1 and ERK2 but also activates another proline-directed kinase that is distinguishable from ERK1/2 based on its strong binding to anion exchange resin and the lack of reactivity with anti-ERK1/2 antibodies (1). We further noted that this kinase readily phosphorylates cPLA 2 but not the S505A mutant of cPLA 2 . This observation indicated that the serine residing within the MAP kinase consensus sequence (i.e. Pro-Leu-Ser 505 -Pro) is the target phosphorylation site for the kinase. Significantly, the thrombin receptor agonist peptide SFLLRN also activated this proline-directed kinase but completely failed to stimulate ERK1/2. Nonetheless SFLLRN, like thrombin, mediated activation of cPLA 2 by phosphorylation, and we reasoned that this unidentified kinase could play a role in the signal transduction pathways activated through the thrombin receptor. We therefore further characterized the kinase with the goal to determine its identity and define its role in the thrombin-induced signaling events during platelet activation. EXPERIMENTAL PROCEDURESPlatelet Isolation and Incubation-Fresh human platelets were prepared from platelet-rich plasma of drug-free volunteers in the presence of prostacyclin (10 Ϫ8 M) and apyrase (0.5 units/ml) as described previously (2), suspended at 1.25 ϫ 10 9 /ml in 140 mM NaCl, 27 mM KCl, 1 mM MgCl 2 , 2.2 mM CaCl 2 , 5.5 mM glucose, 0.2 mM EGTA, 10 mM Hepes, pH 7.4, containing 30 M cyclo(S,S)-Mpr(Har)-GDWP-Pen-NH 2 (where Mpr is mercaptopropionyl, Har is homoarginine, and Pen is penicillamine) (3) (kindly provided by Dr. Robert Scarborough, COR Therapeutics), and incubated at 37°C with 5 units/ml ␣-thrombin (ϳ3500 NIH units/mg, Enzyme Research Laboratories). Reactions were terminated by adding (final concentrations) 1% Triton X-100, 5 mM EGTA, 1 mM DTT, 0.2 mM Na 3 VO 4 , 100 nM microcystin (Life Technologies, Inc.), 100 M leupeptin, 0.2 mg/ml aprotinin, 10 M pepstatin A, 1 mM Pefabloc (Centerchem), and 50 mM ␤-glycerophosphate, pH 7.5. The suspension was then briefly sonicated, centrifuged for 30 min at 100,000 ϫ g using a Sorvall RC M120EX microcentrifuge, and diluted with MonoQ buffer as indicated.Partial Purification of p38 and ERK Kinases by MonoQ Chromatography-High speed supernatants were subjected to chromatography on a MonoQ HR 5/5 column (Pharmacia Biotech Inc.) at a flow rate of 1.5 ml/min ...

The deposition of the ␤ amyloid peptide in neuritic plaques and cerebral blood vessels is a hallmark of Alzheimer's disease (AD) pathology. The major component of the amyloid deposit is a 4.2-kDa polypeptide termed amyloid ␤-protein of 39 -43 residues, which is derived from processing of a larger amyloid precursor protein (APP). It is hypothesized that a chymotrypsin-like enzyme is involved in the processing of APP.We have discovered a new serine protease from the AD brain by polymerase chain reaction amplification of DNA sequences representing active site homologous regions of chymotrypsin-like enzymes. A cDNA clone was identified as one out of one million that encodes Zyme, a serine protease. Messenger RNA encoding Zyme can be detected in some mammalian species but not in mice, rats, or hamster. Zyme is expressed predominantly in brain, kidney, and salivary gland. Zyme mRNA cannot be detected in fetal brain but is seen in adult brain. The Zyme gene maps to chromosome 19q13.3, a region which shows genetic linkage with late onset familial Alzheimer's disease.When Zyme cDNA is co-expressed with the APP cDNA in 293 (human embryonic kidney) cells, amyloidogenic fragments are detected using C-terminal antibody to APP. These co-transfected cells release an abundance of truncated amyloid ␤-protein peptide and shows a reduction of residues 17-42 of A␤ (P3) peptide. Zyme is immunolocalized to perivascular cells in monkey cortex and the AD brain. In addition, Zyme is localized to microglial cells in our AD brain sample. The amyloidogenic potential and localization in brain may indicate a role for this protease in amyloid precursor processing and AD.The generation of the ␤ amyloid peptide is thought to be the result of processing of the amyloid precursor protein (APP) 1 by one or more proteases. After the deduced amino acid sequence of APP was revealed, a number of laboratories initiated studies to purify and characterize the N-terminal cleaving enzyme of amyloid ␤-protein (A␤), termed ␤-secretase (1). The cleavage of the Met 596 -Asp 597 bond of the full-length APP generates the N-terminal amino acid of A␤, which was first shown by Glenner and Wong (2) to be aspartic acid. ␤-Secretase is yet an unidentified protease.Several themes and strategies influenced the direction of investigation of ␤-secretase. The first strategy was to follow a traditional biochemical purification. Assays were utilized in which short peptide substrates were substituted for the large transmembrane precursor protein (1). Any enzyme capable of making a methionine (M)/aspartic acid (D) cleavage could be designated a potential ␤-secretase. The second theme, since the amino acid that surrounded the N terminus of A␤ was found to be a methionine, was some laboratories have rationalized that a search for an enzyme with chymotrypsin-like specificity (a specificity for cleavage of subtrates containing a neutral hydrophobic residue at the S1 subsite) was necessary (3-7).To facilitate the second approach, we have developed a method to identify chymotrypsin-l...

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The results demonstrate the feasibility of non-invasive investigation of placental oxygenation using MRI and the sensitivity of R1 oxygen-enhanced MRI to gestational age. The techniques have the potential to provide unique noninvasive biomarkers in compromised pregnancies.

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These results indicate that both reactive astrocytes and microglia contain elevated levels of cPLA2. Induction of cPLA2 was confined to areas of neurodegeneration and likely precedes its onset. The results suggest that reactive glia may play a role in the pathophysiology of delayed neuronal death after transient global forebrain ischemia.