Responsive microsecond-lifetime photoluminescent probes for analysis of protein kinases and their inhibitors

Responsive ARC-Lum probes were used for measurement of the concentration of active protein kinases (PKs) and determination of affinity of inhibitors of PKs. ARC-Lum probes incorporate thiophene or a selenophene heterocycle and a fluorophore conjugated to the lysine residue in the peptide fragment. In the complex with a PK, ARC-Lum probes emit long-lifetime (microsecond-scale) luminescence at the emission wavelengths of the fluorescent label if the complex is illuminated at the excitation wavelength of the thiophene- or selenophene-containing phosphorescence donors. Bisubstrate ARC-Lum probes bind with sub-nanomolar affinity with several PKs of the AGC group. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Oligonucleotides Containing an Acridine Group Covalently Bonded to the Nucleotide Flanking the 3′-3′ Phosphodiester Junction for Alternate Strand Triple Helix Formation

Abstract

Oligonucleotides with a 3′-3′ inversion of polarity and containing an acridine group attached to nucleotide base flanking the 3′-3′ phosphodiester bon have been synthesized, characterized and used as third strand in alternate triple helix formation. CD melting studies and molecular mechanics calculations have been carried out to investigate these triplex structures.     

Effect of fluorochrome location in protein A on sensing efficiency of IgG

Fragment B of protein A conjugated with fluorescein at various lysine residues is prepared and separated by using a DEAE column in anion-exchange chromatography. The binding of IgG Fc to fragment B contributes to an additional positive electric potential around fragment B. The change in the local electrostatic environment and pH can then be specifically monitored by measuring the fluorescence intensity of fluorescein conjugated with fragment B before and after the introduction of IgG. The studies for the quantitative dependence of fluorescein location on the effectiveness of fluorescein for sensing the protein A-IgG reaction are presented and discussed. (c) 1993 John Wiley & Sons, Inc.     

Internalization of an intact doxorubicin immunoconjugate

An immunoconjugate between doxorubicin and anti-(carcinoembryonic antigen) (CEA) was prepared by using aminodextran (M _r=40 000) as the intermediate carrier, and the carbohydrate moiety of the antibody as the linking site. The resulting immunoconjugate was subjected to an in vitro evaluation for the internalization on the target cells (LoVo), and compared to that of unconjugated antibody, as well as the cellular uptake of unconjugated doxorubicin. The internalization was evaluated microscopically by following the translocation of the red fluorescence of doxorubicin and the green fluorescence of the fluorescein-isothiocyanate-labeled goat anti-(mouse Ig) antibody, which visualizes the location of the primary mouse antibody. Anti-CEA monoclonal antibody (NP-4) was found to internalize into LoVo cells. The immunoconjugate made with this antibody was similarly internalized, and the doxorubicin was found to distribute with the primary antibody. The cell surface and cytoplasm were the major compartments of their distribution. These results indicate that the drug molecules were indeed delivered into the cells by the antibody as an intact conjugate. Unconjugated doxorubicin, on the contrary, was quickly absorbed by the cells and concentrated in the nucleus within 30 min, and never showed a distribution in the cytoplasm or cell membrane as in the nucleus by this procedure. The intermediate drug conjugate, doxorubicin-dextran, did not show internalization. The internalization of NP-4 antibody (or the doxorubicin conjugate) was also confirmed by studying the intracellular catabolism of the cell-bound antibody (or conjugate). The release of the degraded antibody by the cells, as differentiated by trichloroacetic acid precipitation techniques, was considered an indication of internalization. Lysosomes were involved in the degradation, since the process was markedly inhibited in the presence of the lysosomal enzyme inhibitor, ammonium chloride.Supported in part by USPHS grant CA 39841 from the NIH, grant EDT-16 from the American Cancer Society, and grant 89-240360-6 from the New Jersey Commission on Science and Technology.     

Alpha-methylprednisolone conjugated cyclodextrin polymer-based nanoparticles for rheumatoid arthritis therapy

A glycinate derivative of alpha-methylprednisolone (MP) was prepared and conjugated to a linear cyclodextrin polymer (CDP) with a loading of 12.4% w/w. The polymer conjugate (CDP-MP) self-assembled into nanoparticles with a size of 27 nm. Release kinetics of MP from the polymer conjugate showed a half-life (t1/2) of 50 h in phosphate buffer solution (PBS) and 19 h in human plasma. In vitro, the proliferation of human lymphocytes was suppressed to a similar extent but with a delayed effect when CDP-MP was compared with free MP. In vivo, CDP-MP was administered intravenously to mice with collagen-induced arthritis and compared with free MP. CDP-MP was administered weekly for six weeks (0.07, 0.7, and 7 mg/kg/week) and MP was administered daily for six weeks (0.01, 0.1, and 1 mg/kg/day). Body weight changes were minimal in all animals. After 28 days, a significant decrease in arthritis score was observed in animals treated weekly with an intermediate or high dose of CDP-MP. Additionally, dorsoplantar swelling was reduced to baseline in animals treated with CDP-MP at the intermediate and high dose level. Histological evaluation showed a reduction in synovitis, pannus formation and disruption of architecture at the highest dose level of CDP-MP. MP administered daily at equivalent cumulative doses showed minimal efficacy in this model. This study demonstrates that conjugation of MP to a cyclodextrin-polymer may improve its efficacy, leading to lower doses and less frequent administration for a safer and more convenient management of rheumatoid arthritis.     

Synthetic peptides as carriers for cellular import of drugs

We describe the solid phase synthesis of anamphipathic peptide C-terminated by a cysteamide groupwhich allows further addition after removal from theresin and cleavage of the side-chain protectinggroups. The peptide is shown to be rapidlyinternalized by cells with a nuclear localization ofthe peptide. When the peptide is linked to anoligonucleotide, the conjugate is also internalizedwith a final localization that is mainly cytoplasmic.     

The Evaluation of Roadside Surveys of Blood Alcohol Content in Drivers in the Absence of Correlative Information

An evaluation methodology is outlined for roadside surveys of blood alcohol content in drivers, where correlative attributes are absent or at most weakly related to the alcohol levels. The methodology is based on concepts of statistical information theory, and may be extended into a continuous model of the distribution systems arising from such or similar surveys with voluntary participation.