Bassem Y. Azizeh

Molecular dating of senile plaques in the brains of individuals with Down syndrome and in aged dogs.

beta-Amyloid (Abeta) is a constituent of senile plaques found with increasing age in individuals with Down syndrome (DS) and in the canine model of aging. Sections of DS and dog brain were immunostained using an affinity-purified polyclonal antibody for a posttranslationally modified Abeta with a racemized aspartate at position 7 (d7C16). The immunostaining characteristics of d7C16 Abeta in DS and dog brain indicate that it is present in all plaque subtypes, including the thioflavin-S-negative diffuse plaques that develop with age in dogs. The youngest DS case exhibited weak immunolabeling for d7C16 but the extent of d7C16-positive plaques increased with age. In addition, d7C16-positive plaques were initially found in clusters in the superficial layers of the frontal and entorhinal cortex but, with advancing age, increasing numbers appeared in deeper layers, suggesting a progression of Abeta deposition from superficial to deeper cortical layers. Ultrastructural studies in DS brain were confirmed using perfused dog brain and provided consistent results; thioflavin-S-negative diffuse plaques consist of fibrillar Abeta and racemized Abeta is associated with thicker and more highly interwoven fibrils than nonracemized Abeta. The use of antibodies to modified forms of the Abeta protein should provide insight into the progression of plaque pathology in DS and Alzheimers disease brain.

Pure Glucagon Antagonists: Biological Activities and cAMP Accumulation Using Phosphodiesterase Inhibitors

Five new glucagon analogues have been designed, synthesized, characterized and their biological activities tested. The investigation was centered on modifications in the N-terminal region in particular, residues at Thr5, Phe6 and Tyr10 positions, with the goal of obtaining pure glucagon antagonists in our newly developed high sensitivity cAMP accumulation assay. The structures of the designed compounds are: [des-His1, des-Phe6, Glu9] glucagon-NH2 (1); [des-His1, des-Phe6, Glu9, Phe10]glucagon-NH2 (2); [des-His1, Tyr5, des-Phe6, Glu9]glucagon-NH2 (3); [des-His1, Phe5, des-Phe6, Glu9]glucagon-NH2 (4) and [des-His1, des-Phe6, Glu9, D-Arg18]glucagon-NH2 (5). The binding potencies IC50 values in (nM) were 48.0, 27.4, 26.0, 20.0 and 416.0, respectively. All of these analogues when tested in the classical adenylate cyclase assay demonstrate antagonist properties, and in competition experiments, all caused a rightward-shift of the glucagon stimulated adenylate cyclase dose-response curve. The pA2 values for these analogues were 8.20 (1); 6.25 (2); 6.10 (3); 6.25 (4); and 6.08 (5), respectively. A newly revised assay has been developed to determine the intracellular cAMP accumulation levels in hepatocytes at the highest possible sensitivity. Four of the five glucagon analogues in this report (analogues 1, 2, 4 and 5), did not activate the adenylate cyclase in the presence of Rolipram up to a maximal physiological concentration of 1 microM, and thus are pure antagonists.

Cross-linking of NCAM receptors on neurons induces programmed cell death

Programmed cell death has been implicated in the loss of neurons that occurs in many neurodegenerative diseases. This has led to an increased interest in the types of stimuli that can initiate neurons to undergo programmed cell death. Previously, we have shown that cross-linking of membrane receptors with the lectin concanavalin A can trigger programmed cell death in neurons [D.H. Cribbs, V.M. Kreng, A.J. Anderson, C.W. Cotman, Cross-linking of Concanavalin A receptors on cortical neurons induces programmed cell death, Neuroscience 75 (1996) 173-185]. Concanavalin A, however, binds to many surface glycoproteins and therefore, it is important to determine whether certain specific receptors can initiate the program. We found that surface immobilized anti-neural cell adhesion molecules (NCAM) monoclonal antibodies provide a good substrate for adhesion and neurite outgrowth for cortical neurons. However, neurons treated directly with soluble anti-NCAM monoclonal antibodies show significant cell death after 24 h and exhibit the morphological and biochemical features indicative of apoptosis, including membrane blebbing, cell shrinkage, condensation of nuclear chromatin and internucleosomal DNA cleavage.

[des His1, des Phe6, Glu9]glucagon amide: A newly designed "pure" glucagon antagonist

Abstract We report the synthesis and biological activity of a new glucagon analog that was designed as a glucagon receptor antagonist by appropriate modifications in the N-terminal region of glucagon. The structure of the new analog is [des His 1 , des Phe 6 , Glu 9 ]glucagon amide , and its binding potency IC 50 value of 48 nM. The compound was found to be a pure antagonist in a new much more sensitive assay for glucagon stimulated cAMP accumulation activity and showed a pA 2 value of 8.20 in this assay. We report the sythesis and biological activity of a new glucagon analog that was designed as a glucagon receptor antagonist. The new analog, [ des His 1 , des Phe 6 , Glu 9 ]glucagon, amide , was found to be a pure antagonist in a new more sensitive assay for partial agonist activity, with a binding potency IC 50 of 48 nM and a pA 2 valueof 8.20.