Background and Purpose-Delayed neuronal death is a hallmark feature of stroke and the primary target of neuroprotective strategies. Caspase-independent apoptosis pathways are suggested as a mechanism for the delayed neuronal injury. Here we test the hypothesis that one of the caspase-independent apoptosis pathways is activated by BNIP3 and mediated by EndoG. Methods-We performed immunohistochemistry, Western blotting, cell transfection, subcellular fractionation, and RNA interfering to analyze the expression and localization of BNIP3 and EndoG in degenerating neurons in models of stroke and hypoxia. Results-BNIP3 was upregulated in brain neurons in a rat model of stroke and in cultured primary neurons exposed to hypoxia. The expressed BNIP3 was localized to mitochondria. Both forced expression of BNIP3 by plasmid transfection and induced expression of BNIP3 by hypoxia in neurons resulted in mitochondrial release and nuclear translocation of EndoG and neuronal cell death. Knockdown of BNIP3 by RNAi inhibited EndoG translocation and protected against hypoxia-induced neuronal death. Conclusions-BNIP3 plays a role in delayed neuronal death in hypoxia and stroke and EndoG is a mediator of the BNIP3-activated neuronal death pathway. The results suggest that BNIP3 may be a new target for neuronal rescue strategies.

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Optical imaging in the near-infrared (NIR) range enables detecting ligand-receptor interactions and enzymatic activity in vivo due to lower scattering and absorption of NIR photons in the tissue. We designed and tested prototype NIR fluorescent oligodeoxyribonucleotide (ODN) reporters that can sense transcription factor NF-B p50 protein binding. The reporter duplexes included donor NIR Cy5.5 indodicarbocyanine fluorochrome linked to the 3 end of the first ODN and NIR acceptor fluorochromes (indodicarbocyanine Cy7 or, alternatively, a heptamethine cyanine IRDye 800CW) that were linked at the positions ؉8 and ؉12 to the complementary ODN that encoded p50 binding sites. Both Cy7 and 800CW fluorochromes were linked by using hydrophilic internucleoside phosphate linkers that enable interaction between the donor and the acceptor with no base-pairing interference. We observed efficient fluorescence resonance energy transfer (FRET) both in the case of Cy5.5-Cy7 and Cy5.5-800CW pairs of fluorochromes, which was sensitive to the relative position of the dyes. Higher FRET efficiency observed in the case of Cy5.5-Cy7 pair was due to a larger overlap between the ODN-linked Cy5.5 emission and Cy7 excitation spectra. Fluorescent mobility shift assay showed that the addition of human recombinant p50 to ODN duplexes resulted in p50 binding and measurable increase of Cy5.5 emission. In addition, p50 binding provided a concomitant protection of FRET effect from exonuclease-mediated hydrolysis. We conclude that NIR FRET effect can be potentially used for detecting protein-DNA interactions and that the feasibility of detection depends on FRET efficacy and relative fluorochrome positions within ODN binding sites.carbocyanine ͉ transcription factor

As the number of diagnostic and therapeutic applications utilizing gold nanoparticles (AuNPs) increases, so does the need for AuNPs that are stable in vivo, biocompatible, and suitable for bioconjugation. We investigated a strategy for AuNP stabilization that uses methoxypolyethylene glycol-graft poly-l-lysine copolymer (MPEG-gPLL) bearing free amino groups as a stabilizing molecule. MPEG-gPLL injected into water solutions of HAuCl4 with or without trisodium citrate resulted in spherical (Zav = 36 nm), monodisperse (PDI= 0.27), weakly positively charged nanoparticles (AuNP3) with electron-dense cores (diameter: 10.4±2.5 nm) and surface amino groups that were amenable to covalent modification. The AuNP3 were stable against aggregation in the presence of phosphate and serum proteins and remained dispersed after their uptake into endosomes. MPEG-gPLL-stabilized AuNP3 exhibited high uptake and very low toxicity in human endothelial cells, but showed a high dose-dependent toxicity in epithelioid cancer cells. Highly stable radioactive labeling of AuNP3 with 99mTc allowed imaging of AuNP3 biodistribution and revealed dose-dependent long circulation in the blood. The minor fraction of AuGNP3 was found in major organs and at sites of experimentally induced inflammation. Gold analysis showed evidence of a partial degradation of the MPEG-gPLL layer in AuNP3 particles accumulated in major organs. Radiofrequency-mediated heating of AuNP3 solutions showed that AuNP3 exhibited heating behavior consistent with 10 nm core nanoparticles. We conclude that PEG-pPLL coating of AuNPs confers “stealth” properties that enable these particles to exist in vivo in a non-aggregating, biocompatible state making them suitable for potential use in biomedical applications such as non-invasive radiofrequency cancer therapy.

We are investigating a novel pretargeting approach involving an initial IV injection of antitumor antibody conjugated with a phosphorodiamidate morpholino oligomer (MORF, a DNA analog) and the subsequent IV injection of the radiolabeled complement oligomer (cMORF). In this paper, the cMORF was labeled with 188 Re using MAG 3 as chelator for therapeutic applications. Since (c) MORFs are unstable in acidic condition, an optimal labeling pH was first selected and the other labeling factors were then examined. A labeling efficiency of greater than 90% can be achieved even at a concentration of MAG 3 -cMORF as low as 0.8 μM. The labeled cMORF is stable and capable of hybridizing to its complement.

Purpose: Pretargeting has been attracting increasing attention as a drug delivery approach.We recently proposed Watson-Crick pairing of phosphorodiamidate morpholino oligomers (MORF) for the recognition system in tumor pretargeting. MORF pretargeting involves the initial i.v. injection of a MORF-conjugated antitumor antibody and the subsequent i.v. injection of the radiolabeled complement. Our laboratory has reported on MORF pretargeting for diagnosis using 99m Tc as radiolabel.We now report on the use of MORF pretargeting for radiotherapy in a mouse tumor model using 188