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Variations in fluid viscosity are linked to a variety of functions and diseases both at the cellular level (e.g., membrane and cytoplasmic viscosity changes in cell signaling modulation) 1 and at the organismal level (e.g., blood, plasma, or lymphatic fluid viscosity changes in diabetes, hypertension, infarction, and aging). 2 It has been proposed that monitoring of biofluid viscosity could provide a diagnostic tool for the detection of diseases. 3 Since mechanical devices do not provide the spatial and temporal resolution needed, a new type of fluorescent-based viscosity sensors was developed. 4 These sensors are based on a class of environmentsensitive fluorescent dyes that are characterized by a viscositydependent emission quantum yield. 4,5 The chemical structure of these dyes contains an electron donor unit (such as a nitrogen atom) in conjugation with an electron acceptor unit (such as a nitrile). Upon photoexcitation, the two units can rotate relative to each other in a manner that is dependent on the viscosity of their environment. Representative examples of such fluorescent rotors are 9-(dicyanoVinyl)julolidine (DCVJ, 1) and 2-cyano-3-(4-dimethylaminophenyl)acrylic acid methyl ester (CMAM, 2) ( Figure 1). 5 Their viscosity-dependent fluorescent quantum yield is described by the Förster-Hoffmann equation (eq 1). 6 Fluorescent molecular rotors have been used for viscosity studies that are performed by steady-state fluorescence through emission intensity measurements. This method suffers, however, from drawbacks arising from changes of the fluid optical properties and fluctuations in dye concentrations. An additional disadvantage is that a calibration curve is needed for the absolute determination of viscosity. 5 As a consequence, changes in fluid properties and dye concentration may cause erroneous readings.We hypothesized that a dual dye composed of two distinct fluorescent units, one providing an internal intensity reference and the other acting as a viscosity sensor, would create a ratiometric sensing system, thus overcoming the above disadvantages. Dividing the sensor emission intensity by the reference emission intensity would yield a normalized intensity that should not only eliminate some of the fluid-and concentration-related artifacts but also provide a means to quantify viscosity by an internal reference. To test this hypothesis, we synthesized a compound 4 in which the CMAM motif was coupled with 7-methoxycoumarin-3-carboxylic acid (MCCA, 3). We chose MCCA as the donor fluorophore in order to induce excitation of the rotor moiety (CMAM) via Resonance Energy Transfer (RET). 7 We envisioned that, due to its viscosity-independent quantum yield, MCCA could be used as both the internal reference and the RET donor. The latter event could then excite the CMAM motif, resulting in a viscosity-dependent emission of the rotor. The linker was chosen to maintain a distance between the chromophores in the same range as the Förster distance 7a to allow considerable energy transfer to the acceptor combined w...

ErbB‐2 is overexpressed in several human cancers and conveys a transforming activity that is dependent on tyrosine kinase activity. Antibodies and T cells to ErbB‐2 have been isolated from cancer patients, indicating ErbB‐2 as a potential target of active vaccination. In this study, 3 mutant ErbB‐2 DNA constructs encoding full‐length, ErbB‐2 proteins were tested as tumor vaccines. To eliminate tyrosine kinase activity, the ATP binding lysine residue 753 was substituted with alanine by replacing codon AAA with GCA in mutant ErbB‐2A. To direct recombinant ErbB‐2 to the cytoplasm where major histocompatibility complex (MHC) I peptide processing takes place, the endoplasmic reticulum (ER) signal sequence was deleted in cyt ErbB‐2. The third construct cyt ErbB‐2A contained cytoplasmic ErbB‐2 with the K to A mutation. Expression of recombinant proteins was measured by flow cytometry in transfected murine mammary tumor cell line D2F2. Transmembrane ErbB‐2 and ErbB‐2A were readily detected. Cytoplasmic ErbB‐2 and ErbB‐2A were detected only after the transfected cells were incubated overnight with a proteasome inhibitor, indicating prompt degradation upon synthesis. ErbB‐2 autophosphorylation was eliminated by the K to A mutation as demonstrated by Western blot analysis. Growth of ErbB‐2‐positive tumor in BALB/c mice was inhibited after vaccination with ErbB‐2 or ErbB‐2A, but not with cyt ErbB‐2 or cyt ErbB‐2A. ErbB‐2A that is free of tyrosine kinase activity is a potential candidate for anticancer vaccination. The 3 mutant constructs should be useful tools to delineate the role of individual immune effector cell in ErbB‐2‐specific antitumor immunity and to develop strategies for enhancing such immunity. Int. J. Cancer 81:748–754, 1999. © 1999 Wiley‐Liss, Inc.

It has been shown that compounds containing the p-N,N,-dialkylaminobenzylidene cyanoacetate motif can serve as fluorescent non-mechanical viscosity sensors. These compounds, referred to as molecular rotors, belong to a class of fluorescent probes that are known to form twisted intramolecular charge-transfer complexes in the excited state. In this study we present the synthesis and spectroscopic characterization of these compounds as viscosity sensors. The effects of the molecular structure and electronic density of these rotors to the emission wavelength, fluorescence intensity and viscosity sensitivity are discussed.