No abstract

Abelson tyrosine kinase (Abl) is a non-receptor tyrosine kinase which is frequently coupled with adaptor proteins to interact with its substrates for the regulation of cytoskeleton rearrangement, cell growth and apoptosis in response to a variety of biological stimuli. The Abl interactor (Abi) family members were first identified as adaptor proteins of Abl for regulating Abl transforming and kinase activity. In the present study, we used a yeast two-hybrid screen to identify Cdc2 as a novel Abi-binding protein. This finding led us to investigate the role of Abi in linking Abl and Cdc2. These three proteins formed a trimeric complex in Drosophila and mammalian cells. The expression of Abi in cells greatly enhanced the formation of the Abl-Cdc2 complex, suggesting that Abi functions as an adaptor protein facilitating the binding between Abl and Cdc2. We show that Abi promotes Abl-mediated phosphorylation of Cdc2 at tyrosine 15 and inactivation of Cdc2 kinase activity. Furthermore, coexpression of Abl and Abi in Drosophila S2 cells led to suppression of cell growth. These data suggest that Abl signaling may be involved in the downregulation of Cdc2 kinase in cell cycle control.

Among the miajor obstacles to clarifying molecular mechanisms involved in amyloid metabolism in Alzheimer disease has been the unavailability oflaboratory models for this uniquely human disorder. The present studies were aimed at establishing genetically engineered cell lines that overexpress amyloid immunoreactivity and that may be relevant to amyloid accumulation in the Alzheimer disease brain. We used cloned amyloid cDNA that contains a region encoding A4 (Iipolypeptide) amino acids along with recently developed tumor virus vectors derived from simian virus 40 to prepare transformed cells. After transient and permanent transfection, a variety of cell types overexpressed A4 immunoreactivity that was detected by highly specific monoclonal antibodies. We observed that the use of an amyloid subdomain containing the A4 region, but lacking the sequence of a Kunitz-type protease inhibitor found in amyloid precursor protein variants, was sufficient to obtain cells that overproduced an A4 epitope. The transformed cells were readily propagated in culture and may provide an experimental medium to elucidate aspects of the molecular pathogenesis of Alzheimer disease. The cellular models may also serve as tools for deriving potentially useful therapeutic agents.cDNA for the amyloid precursor protein (APP) has been prepared and sequenced from mRNA of the fetal brain (1, 2), from the nondemented adult brain (3, 4), and directly from the Alzheimer disease (AD) brain (5). In the latter study, we showed that the genetic transcript of the /-amyloid (6), or A4 domain (1), as well as flanking nucleotides that comprise approximately half the entire precursor structure, has the same sequence as the fetal transcript (1,5). Thus, cellular regulatory factors that affect processing at the A4 site, rather than a specific A4 peptide structure, may be involved in the overaccumulation of amyloid in AD. However, because AD is a uniquely human disorder for which neither cellular nor animal models have existed, there are limits upon direct biological studies.Therefore, a cellular model for amyloidosis that may be relevant to AD would be useful to distinguish among various hypotheses related to amyloid overaccumulation. The present work was aimed at determining whether or not cloned amyloid cDNA containing the A4 domain, linked to a suitable vector, could be used to transfect host cells to overexpress the A4 peptide at immunologically detectable levels. It was not immediately clear whether available transfection methods would be applicable to our goals because of the possibility that amyloid overproduction would lead to a potentially lethal mutation. Alternatively, amyloid protein might be rapidly degraded soon after expression and would consequently not be detectable by immunologic procedures; this possibility was strengthened by recent reports indicating that a variety of cultured cell lines contain amyloid precursor variants with internal protease inhibitor sequences (7-9).A portion of the data was reported previously in preliminary ...

To gain insight into factors associated with the excessive accumulation of fi-amyloid in the Alzheimer disease (AD) brain, the present studies were initiated to distinguish between a unique primary structure of the ADspecific amyloid precursor mRNA vis a vis other determinants that may affect amyloid levels. Previous molecular cloning experiments focused on amyloid derived from sources other than AD cases. In the present work, we cloned and characterized amyloid cDNA derived directly from AD brain mRNA. Poly(A) + RNA from AD cortices was used for the preparation of Agtll recombinant cDNA libraries. An insert of 1564 nucleotides was isolated that included the (1-amyloid domain and corresponded to 75% of the coding region and :70% of the 3'-noncoding region of the fetal precursor amyloid cDNA reported by others. On RNA blots, the AD amyloid mRNA consisted of a doublet of 3.2 and 3.4 kilobases. In control and AD cases, the amyloid mRNA levels were nonuniform and were independent of glial-specific mRNA levels. Based on the sequence analysis data, we conclude that a segment of the amyloid gene is expressed in the AD cortex as a high molecular weight precursor mRNA with major coding and 3'-noncoding regions that are identical to the fetal brain gene product.

Pamamycin was previously identified as a stimulator of aerial mycelium formation in Streptomyces alboniger and as a new antibiotic. Studies in Staphylococcus aureus grown in brain heart infusion broth showed that this antibiotic was bacteriostatic at 0.1 to 0.3 U/ml and bactericidal at 0.5 U/ml or higher. At concentrations which inhibited growth ca. 40%, pamamycin inhibited the uptake of nucleosides and Pi, as well as purine and pyrimidine bases, and the incorporation of these precursors into nucleic acids. Under the same conditions, the antibiotic had no effect on protein and cell wall synthesis, glucose utilization, or the uptake of amino acids, 2-deoxyglucose, or Mn2+. Further studies suggested that the primary action of pamamycin was on transport processes rather than de novo ribonucleic acid and deoxyribonucleic acid synthesis. Direct in vitro studies of nucleoside transport with isolated membrane vesicles confirmed this conclusion. Pamamycin bound tightly to bacterial membranes and induced significant release of ultraviolet-absorbing material at bactericidal concentrations. It was concluded that the mechanism of growth inhibition involves alteration of membrane-associated cellular functions. Inhibition of Pi transport is a likely primary target for this effect.The streptomycetes are a unique group of procaryotic organisms. They not only produce many biologically active compounds, such as antibiotics, but also have a definitive developmental cycle (7). There are several reports on the control and regulation of the formation of spores and aerial mycelium by endogenous factors isolated from streptomycetes (8,9,25). The mechanism of action of those factors is still unknown (23,24,26 which inhibits the transport of nucleosides, purine and pyrimidine bases, and Pi.MATERIALS AND METHODS Pamamycin. The isolation, purification, and characterization of this new antibiotic have been described previously (15). Antibiotic purified through the silicic acid column chromatography step was used in this study. One unit of pamamycin gave a 17-mm zone of inhibition against Sarcina lutea on tryptic soy agar.Stocks were stored in toluene (0.5 to 1 U/pl) at 4°C.The desired amount needed for an experiment was dried under nitrogen and redissolved in dimethyl sulfoxide (DMSO) unless otherwise indicated. This solvent had no effect in any of the experiments reported.Bacteria and media. S. aureus ATCC 6538P, S.