MAB349 Sigma-AldrichAnti-Amyloid Precursor Protein Antibody, A4(695), clone 1.D5

We tested the independent utility of β-amyloid precursor protein (β-APP) immunohistochemical staining as evidence of brain trauma in the deaths of young children. Blinded reviewers retrospectively reviewed immunostained brain tissues from homicidal deaths, age-matched control cases without evidence of trauma, as well as cases of sudden infant death syndrome (SIDS). The reviewers correctly identified five of the seven cases with documented inflicted head trauma. However, one of seven age-matched control cases and one of 10 SIDS/sudden unexplained death in infancy (SUDI) cases demonstrated staining patterns similar to those seen in cases of inflicted trauma. We discuss these cases and the circumstances surrounding them with the intent to explain the difficulties associated with immunohistological interpretation of axonal injury. Although the utility of β-APP is quite powerful if not confounded by global hypoxic-ischemic injury, ultimately, β-APP studies should be only one piece of information in the determination of cause and manner of death.

Multilayered fibroblast sheets have applications as cell transplants for tissue engineering. One way to increase their therapeutic efficacy is to increase cell numbers in a graft, but the factors influencing multilayered growth remain poorly understood. In this study, we investigated the roles of focal adhesion (FA) assembly and intercellular cohesion through fibronectin (FN) in the proliferation of normal human fibroblasts at confluence. Density-dependent growth-arrested fibroblasts resumed DNA synthesis when cultured in multilayer formation medium (MFM) containing transforming growth factor-beta1, ascorbic acid, and serum. This proliferation depended on alpha 5 beta 1-integrin-mediated cell-FN-cell interactions because blocking them with antibodies inhibited DNA synthesis. However, cell-FN-cell cohesion operated well regardless of exposure to MFM, judging from several parameters, including FN matrix deposition, activated beta1 integrin expression, and stress fiber development. Density-arrested cells formed few FAs at the cell center. Exposure of the cells to MFM induced the formation of vinculin-, paxillin-, and phosphotyrosine-containing FAs throughout the ventral cell-surface, indicating ROCK-mediated actomyosin contractile force generation. When the assembly of FAs was inhibited with either the ROCK inhibitor Y-27632 or the myosin II inhibitor blebbistatin, the up-regulation of DNA synthesis by MFM was suppressed. The drugs did not impair FN matrix deposition, activated beta1 integrin expression, and stress fiber development. Thus, these results indicate that the formation of FAs promotes the proliferation of confluent fibroblasts with the support of alpha 5 beta 1-integrin-mediated cell-FN-cell cohesion. The present findings provide insights into the rational design of high-density fibroblast transplants.

The abuse of drugs such as opioids and 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy') can have detrimental effects on the cognitive functions, but the exact molecular mechanism whereby these drugs promote neurodegeneration remains to be elucidated. The major purpose of the present pilot study was to determine whether the chronic in-vivo administration of morphine (10 mg/kg) or MDMA (1 mg/kg) to rats can alter the expression and processing of amyloid precursor protein (APP), the central molecule in the proposed pathomechanism of Alzheimer's disease. MDMA treatment significantly decreased the production of APP in the cytosolic fraction of the brain cortex. A concomitant 25% increase was found both in the beta-secretase (BACE) and APP mRNA levels (108%). In contrast, in the applied single dosage chronic morphine treatment did not influence either the APP and BACE protein levels or the APP mRNA production. These results indicate that the chronic use of 'ecstasy', but not morphine, may be harmful via a novel mode of action, i.e. by altering the APP expression and processing in the brain.

To study the putative precursor proteins (PreA4(695), PreA4(751), and PreA4(770] of Alzheimer's disease A4 amyloid protein, polyclonal and monoclonal antibodies were raised against a recombinant bacterial PreA4(695) fusion protein. These antibodies were used to identify the precursors in different cell lines as well as in human brain homogenates and cerebrospinal fluid (CSF). The precursors are tyrosine-sulfated, O- and N-glycosylated membrane proteins and have half-lives of 20-30 min in cells. Cells express the polypeptides at their surface but also secrete C-terminal truncated proteins into the medium. These proteins are also found in CSF of both Alzheimer's disease patients and normal individuals. The proteins are derived from their cognate membrane-associated forms by proteolysis and have apparently lost the cytoplasmic and the transmembrane domains. Since the latter contributes to the A4 amyloid sequence, it seems possible that this proteolytic cleavage represents the first step in the formation of A4 amyloid deposits.