Biochemistry
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Cemadotin (LU103793) (NSC D-669356) is a water-soluble synthetic analogue of dolastatin 15 that inhibits cell proliferation in vitro and the growth of human tumor xenografts. Cemadotin is in phase II clinical trials as a promising cancer chemotherapeutic agent. The drug blocks cells at mitosis. ⋯ It reduced the rate and extent of growing and shortening, increased the rescue frequency, and increased the percentage of time the microtubules spent in an attenuated or paused state, neither growing nor shortening detectably. At the lowest effective cemadotin concentrations, dynamics were suppressed in the absence of significant microtubule depolymerization. The results suggest that cemadotin exerts its antitumor activity by suppressing spindle microtubule dynamics through a distinct molecular mechanism by binding at a novel site in tubulin.
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Progressive cerebral deposition of the amyloid beta-protein (Abeta) is believed to play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The highly amyloidogenic 42-residue form of Abeta (Abeta42) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Abeta42 in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Abeta. ⋯ When the same fractions were analyzed by enzyme-linked immunosorbent assays for Abetatotal and Abeta42, Abeta42 was the major Abeta species in the ER fraction (Abeta42:Abetatotal ratio 0.5-1.0), whereas absolute levels of both Abeta42 and Abeta40 were higher in the Golgi fraction and the Abeta42:Abetatoal ratio was 0.05-0.16 there. Mutant PS1 significantly increased Abeta42 levels in the Golgi fraction. Our results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper gamma-secretase processing of APP CTFs, and that PS1 mutations augment Abeta42 levels principally in Golgi-like vesicles.
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The interactions of simian virus 40 (SV40) large T antigen with DNA carrying the viral origin of DNA replication, as well as its interactions with cellular replication proteins, have been investigated by using fluorescent ATP analogues as specific probes. The enhanced fluorescence of 3'(2')-O-(2,4, 6-trinitrophenyl)adenosine diphosphate (TNP-ADP) induced by T antigen binding to the nucleotide was decreased upon binding of T antigen to origin DNA. Similarly, the enhanced fluorescence induced by T antigen binding to TNP-ADP or TNP-ATP was decreased upon binding to human DNA polymerase alpha-primase (pol alpha), but not to replication protein A (RPA). ⋯ The results support a model in which double hexamer assembly on the viral origin occurs by successive binding of 12 free T antigen or monomeric T-nucleotide complexes to the DNA. In contrast with this stepwise assembly of T antigen monomers on DNA, hexameric T antigen was able to bind directly to pol alpha with concomitant release of the bound TNP nucleotide. The possible implications of these results for the mechanism of initiation of SV40 DNA replication are discussed.
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Sphingosine 1-phosphate (SPP), a lipid second messenger formed by the action of sphingosine kinase, has been implicated in regulating diverse biological processes, including growth, survival, and differentiation. N,N-Dimethylsphingosine (DMS) inhibits sphingosine kinase and has been used to investigate the biological roles of SPP; however, little is known of the mechanism of inhibition of sphingosine kinase by DMS. In addition, DMS has been shown to inhibit protein kinase C in vitro. ⋯ DMS also effectively increases cellular levels of ceramide in a variety of cell types, and resetting of the ceramide/SPP rheostat may account for the pro-apoptotic effects of DMS. Moreover, DMS, at concentrations which effectively inhibit sphingosine kinase, has no effect on protein kinase C activity or its membrane translocation. Thus, DMS acts as a specific competitive inhibitor of sphingosine kinase in diverse cell types and is a useful tool to elucidate the role of SPP as an intracellular second messenger.
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The ferritin IRE, a highly conserved (96-99% in vertebrates) mRNA translation regulatory element in animal mRNA, was studied by molecular modeling (using MC-SYM and DOCKING) and by NMR spectroscopy. Cobalt(III) hexammine was used to model hydrated Mg2+. IRE isoforms in other mRNAs regulate mRNA translation or stability; all IREs bind IRPs (iron regulatory proteins). ⋯ U base pair, formed a pocket suitable for Co(III) hexammine binding. On the basis of the effects of Co(III) hexammine on the 1H NMR spectrum and results of automatic docking into the IRE model, the IRE bound Co(III) hexammine at the pocket in the major groove; Mg2+ may bind to the IRE at the same site on the basis of an analogy to Co(III) hexammine and on the Mg2+ inhibition of Cu-(phen)2 cleavage at the site. Distortion of the IRE helix by the internal loop/bulge near a conserved unpaired C required for IRP binding and adjacent to an IRP cross-linking site suggests a role for the pocket in ferritin IRE/IRP interactions.