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PDF file - 546K, In vitro characterization of NTR/CytoCy5S.

PDF file - 99K, Materials and methods of CytoCy5S synthesis, purification, spectral and structural characterization in addition to NTR/CytoCy5S enzymatic reaction conditions and western blots.

The ability to visualize reporter gene expression in vivo has revolutionized all facets of biologic investigation and none more so than imaging applications in oncology. Near-infrared reporter gene imaging may facilitate more accurate evaluation of chemotherapeutic response in preclinical models of orthotopic and metastatic cancers. We report the development of a cell permeable, quenched squarine probe (CytoCy5S), which is reduced by Escherichia coli nitroreductase (NTR), resulting in a near-infrared fluorescent product. Time-domain molecular imaging of NTR/CytoCy5S reporter platform permitted noninvasive monitoring of disease progression in orthotopic xenografts of disseminated leukemia, lung, and metastatic breast cancer. This methodology facilitated therapeutic evaluation of NTR gene–directed enzymatic prodrug therapy with conventional metronidazole antibiotics. These studies show NTR/CytoCy5S as a near-infrared gene reporter system with broad preclinical and prospective clinical applications within imaging, and gene therapy, of cancer. Cancer Res; 73(4); 1276–86. ©2012 AACR.

PDF file - 358K, In vitro viability of NB4, NCI-H460 and MDA-MB-231 cells � NTR � CytoCy5S.

PDF file - 99K

PDF file - 402K, Synthesis, mechanism and spectral characteristics of CytoCy5S.

PDF file - 314K, In vivo characterization of NTR/CytoCy5S fluorescence and fluorescence lifetime distributions in NB4NTR+GFP+ subcutaneous tumors, NCI-H460NTR+Luc+GFP+ orthotopic xenografts and gastrointestinal tract.

PDF file - 718K, In vitro viability of NB4 and MDA-MB-231 cells � NTR � MTZ.

PDF file - 1949K, Flow cytometry quantification of NTR/CytoCy5S fluorescence in NB4 cells � NTR � CytoCy5S.

A new europium(III) chelate, which uses the 2,2′∶6′,2′′-terpyridyl chelating group as sensitizer, has been synthesized and its spectral properties investigated. The chelate, which can incorporate an isothiocyanate group for biological labelling, has proved useful in time resolved fluorescence studies and for delayed fluorescence resonance energy transfer (DEFRET).

A pentamethine cyanine dye has been synthesised that is fluorescent when protonated, becoming non-fluorescent upon proton abstraction. The probe has a pKa of 7.5, with observable changes in the fluorescent emission properties across a pH range of 6.0–8.0, therefore providing a useful probe for studying pH in biological media.

A pentamethine cyanine dye has been synthesised that is fluorescent when protonated, becoming non-fluorescent upon proton abstraction. The probe has a pKa of 7.5, with observable changes in the fluorescent emission properties across a pH range of 6.0–8.0, therefore providing a useful probe for studying pH in biological media.

Cells are frequently interconnected by tunneling nanotubules, recently discovered cylindrical structures which facilitate material exchange. We employed here single molecule fluorescence microscopy to study the diffusion of the glycosylphosphatidylinositol-protein CD59 in the plasma membrane of tunneling nanotubules in living cells at subwavelength resolution. Our study provides the nanotubule radius in vivo, yielding a surprisingly narrow size distribution (7nm standard deviation) around a mean value of 65nm. Moreover, by separating longitudinal and transverse mobilities, we find isotropic diffusion behavior.

Publisher Summary Two distinct high-affinity, membrane-bound cAMP phosphodiesterases can be isolated from rat liver. These are a peripheral plasma membrane enzyme that is activated by insulin alone and the dense-vesicle enzyme that is activated by both insulin and glucagon through apparently distinct mechanisms. Both procedures yield preparations exhibiting a single protein band that comigrated with phosphodiesterases (PDE) activity on nondenaturing gels. This chapter describes plasma membrane method using a crude membrane fraction for the extraction of this enzyme. The dense-vesicle enzyme was purified some 2773-fold, over the specific activity of the homogenate from which it was isolated, with an 18% yield. The enzyme, as purified, is a proteolytically clipped version of the native enzyme, as endogenous lysosomal proteases, which are released by the hypotonic shock procedure, affect its solubilization. The solubilized enzyme can now be purified by standard techniques.

The hormone-stimulated ‘dense-vesicle’ cyclic AMP phosphodiesterase was solubilized as a proteolytically ‘clipped’ species, and purified to apparent homogeneity from rat liver with a 2000-3000-fold purification and a 13-18% yield. It appeared to be a dimer (Mr 112,000), of two Mr-57,000 subunits. Solubilization of either a liver or a hepatocyte membrane fraction, with sodium cholate in the presence of the protein inhibitor benzamidine, identified three protein bands which could be immunoprecipitated by a polyclonal antibody raised against the pure enzyme. The major band at Mr 62,000 is suggested to be the native ‘dense-vesicle’ enzyme, having a Mr-5000 extension which serves to anchor this enzyme to the membrane and which is cleaved off during proteolytic solubilization; the Mr-200,000 band is an aggregate of the Mr-62,000 species, and the Mr-63,000 species is possibly a precursor. The purified ‘clipped’ enzyme hydrolysed cyclic AMP with kinetics indicative of apparent negative co-operativity, with a Hill coefficient (h) of 0.43 and limiting kinetic constants of Km1 = 0.3 +/- 0.05 microM, Km2 = 29 +/- 6 microM, Vmax.1 = 0.114 +/- 0.015 unit/mg of protein and Vmax.2 = 0.633 +/- 0.054 unit/mg of protein. It hydrolysed cyclic GMP with Michaelis kinetics, Km = 10 +/- 1 microM and Vmax. = 4.1 +/- 0.2 units/mg of protein. Cyclic GMP was a potent inhibitor of cyclic AMP hydrolysis, with an IC50 (concn. giving 50% inhibition) of 0.20 +/- 0.01 microM-cyclic GMP when assayed at 0.1 microM-cyclic AMP. This enzyme was inhibited potently by several drugs known to exert positive inotropic effects on the heart, was extremely thermolabile, with a half-life of 4.5 +/- 0.5 min at 40 degrees C, and was shown to be distinct from the rat liver insulin-stimulated peripheral-plasma-membrane cyclic AMP phosphodiesterase [Marchmont, Ayad & Houslay (1981) Biochem. J. 195, 645-652].

Two enzymes displaying cyclic GMP-stimulated cyclic AMP phosphodiesterase activity were purified from rat liver to apparent homogeneity: a ‘particulate enzyme’ found as an integral membrane protein associated with the plasma membrane, and a ‘soluble’ enzyme found in the cytosol. The physical properties of these enzymes were very similar, being dimers of Mr 134,000, composed in each instance of two subunits of Mr = 66,000-67,000. Both enzymes showed similar kinetics for cyclic AMP hydrolysis. They are both high-affinity enzymes, with kinetic constants for the particulate enzyme of Km = 34 microM and Vmax. = 4.0 units/mg of protein and for the cytosolic enzyme Km = 40 microM and Vmax. = 4.8 units/mg of protein. In both instances hydrolysis of cyclic AMP appeared to show apparent positive co-operativity, with Hill coefficients (happ.) of 1.5 and 1.6 for the particulate and cytosolic enzymes respectively. However, in the presence of 2 microM-cyclic GMP, the hydrolysis of cyclic AMP obeyed Michaelis kinetics (happ. = 1) for both enzymes. The addition of micromolar concentrations of cyclic GMP had little effect on the Vmax. for cyclic AMP hydrolysis, but lowered the Km for cyclic AMP hydrolysis to around 20 microM in both cases. However, at low cyclic AMP substrate concentrations, cyclic GMP was a more potent activator of the particulate enzyme than was the soluble enzyme. The activity of these enzymes could be selectively inhibited by cis-16-palmitoleic acid and by arachidonic acid. In each instance, however, the hydrolysis of cyclic AMP became markedly more sensitive to such inhibition when low concentrations of cyclic GMP were present. Tryptic peptide maps of iodinated preparations of these two purified enzyme species showed that there was considerable homology between these two enzyme forms.