The green fluorescent protein (GFP) is a widely used reporter in gene expression and protein localization studies. GFP is a stable protein; this property allows its accumulation and easy detection in cells. However, this stability also limits its application in studies that require rapid reporter turnover. We created a destabilized GFP for use in such studies by fusing amino acids 422-461 of the degradation domain of mouse ornithine decarboxylase (MODC) to the C-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike EGFP, was unstable in the presence of cycloheximide and had a fluorescence half-life of 2 h. Western blot analysis indicated that the fluorescence decay of EGFP-MODC-(422-461) was correlated with degradation of the fusion protein. We mutated key amino acids in the PEST sequence of EGFP-MODC-(422-461) and identified several mutants with variable half-lives. The suitability of destabilized EGFP as a transcription reporter was tested by linking it to NFB binding sequences and monitoring tumor necrosis factor ␣-mediated NFB activation. We obtained time course induction and dose response kinetics similar to secreted alkaline phosphatase obtained in transfected cells. This result did not occur when unmodified EGFP was used as the reporter. Because of its autofluorescence, destabilized EGFP can be used to directly correlate gene induction with biochemical change, such as NFB translocation to the nucleus.Because of its easily detected green fluorescence, the green fluorescent protein (GFP) 1 from the jellyfish Aequorea victoria is a widely used reporter in studies of gene expression and protein localization (1-4). GFP fluorescence does not require any substrate or cofactor (5); hence it is possible to use it in many species for live cell detection purposes. The fluorescence of GFPs is dependent on the key sequence Ser-Tyr-Gly (amino acids 65-67). This sequence undergoes spontaneous oxidation to form a cyclized chromophore (6). Enhanced GFP (EGFP) contains mutations of Ser to Thr at amino acid 65 and Phe to Leu at position 64 and is encoded by a gene with humanoptimized codons (7-9). Crystallographic structures of wildtype GFP and the mutant S65T reveal that the GFP tertiary structure resembles a barrel (10, 11). GFP is a single chain polypeptide of 238 amino acids (12). Most of these amino acids form ␤ sheets that are compacted through an antiparallel structure to form the barrel. An ␣-helix containing the chromophore is located inside the barrel, which shields it from the external environment. The compact structure makes GFP very stable under a variety of conditions, including treatment with protease (1). The stability of GFP limits its application in some studies, including transcriptional induction studies.Cellular proteins differ widely in their stabilities. Rapid turnover in proteins is often caused by signals that induce protein degradation. In some cases, the signal is a primary sequence such as the PEST sequence, a sequence possibly correlated with protein degradation (13,14)...

We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a "fluorescent timer" that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.

It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission max ‫؍‬ 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.It is generally accepted that the enormous variety of colors and fluorescent hues displayed by living organisms are determined by chromoproteins and low molecular weight pigments. As a rule, chromoproteins typically require a prosthetic group: a small nonpeptide molecule or metal ion, which binds to the protein and is essential for the chromogenic properties of the protein (1-6).The only known exception to this rule is green fluorescent protein (GFP) 1 from Aequorea victoria (7). In contrast to other naturally occurring fluorescent proteins, the fluorescence of GFP is due entirely to an internal interaction between amino acids within the protein; no other cofactors or prosthetic groups are required. GFP owes its intrinsic fluorescence to a contiguous Ser-Tyr-Gly sequence centrally located within its primary structure. Upon folding, the protein modifies the fluorophoreforming sequence to produce an extended aromatic system (8 -10), which imparts the characteristic green fluorescence to the mature protein. Due to these distinctive properties, GFP has enjoyed extensive use as a biological marker in vivo (11, 12). Recently we described six novel GFP-like fluorescent proteins (FP) from nonbioluminescent Anthozoa species (13). It therefore became clear that GFP-like proteins are not necessarily components of bioluminescent systems but may simply determine fluorescent coloration of animals.In one particular case, we have shown that a GFP-like FP is responsible for the bright green fluorescence of the tentacle tips in the sea anemone Anemonia majano. However, in another sea anemone, Anemonia sulcata, we found that, although the tentacle tips do exhibit an intense purple color they are not significantly fluorescent (Fig. 1). The similarities of the color localization patterns and the close phylogenetic relationship of these two species led us to hypothesize that A. sulcata contains a purple nonfluorescent GFP homolog in its tentacles. In the p...

We recently reported the discovery of AM-8553 (1), a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Continued research investigation of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface, led to the discovery of a one-carbon tethered sulfone which gave rise to substantial improvements in biochemical and cellular potency. Further investigation produced AMG 232 (2), which is currently being evaluated in human clinical trials for the treatment of cancer. Compound 2 is an extremely potent MDM2 inhibitor (SPR KD = 0.045 nM, SJSA-1 EdU IC50 = 9.1 nM), with remarkable pharmacokinetic properties and in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft model (ED50 = 9.1 mg/kg).

The formation of H 2 Ti 3 O 7 nanotubes, nanofibers, nanowires, and nanobelts via alkali hydrothermal synthesis was studied in detail by TEM and HRTEM. The effects of preparation parameters, such as reaction temperature, duration, and cooling process, on the morphologies of the products are clarified. A universal formation mechanism is proposed based on the growth, split, wrapping, and thickening of Na 2 Ti 3 O 7 nanointermediates, which links all kinds of morphologies observed for H 2 Ti 3 O 7 nanoentities.