Spatial inhomogeneities of poly(N-isopropylacrylamide-co-acrylic acid) (NIPA/AAc) gels were investigated by dynamic light scattering as a function of AAc comonomer concentration and temperature. The results showed the following: (1) The ensemble average scattered intensity, 〈I〉E, decreased with increasing AAc comonomer concentration and increased with temperature. (2) Dynamic fluctuations increased with temperature and diverged at the transition temperature, Tc. (3) Above the Θ temperature of NIPA homopolymer in an aqueous solution, the intensity-intensity time correlation function did not fit a single-exponential function, but fit a double exponential or a stretched exponential, indicating a heterogeneous structure, i.e., polymer-rich and polymer-poor phases at temperatures above Θ.

We have discovered that 3,3',5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC(50) of ~1 μm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser(139))histone H2AX induction and cell growth inhibition was observed.

SummaryWe developed a split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts. In this assay, the N-and C-terminal fragments of Renilla reniforms luciferase are translationally fused to bait and prey proteins, respectively. When the proteins interact, split luciferase becomes activated and emits luminescence that can be measured by a microplate luminometer. Split luciferase activity was measured by first transforming protoplasts with a DNA vector in a 96-well plate. DNA vector expressing both bait and prey genes was constructed through two independent in vitro DNA recombinant reactions, Gateway and CreloxP. As proof of concept, we detected the protein-protein interactions between the nuclear histones 2A and 2B, as well as between membrane proteins SYP (syntaxin of plant) 51 and SYP61, in Arabidopsis protoplasts.

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