The effect of lipid ordering on the kinetics and extent of metarhodopsin II (meta II) formation was evaluated in bovine rhodopsin which had been reconstituted into phosphatidylcholine vesicles containing 0, 15, and 30 mol% cholesterol. The rate of establishment of the dynamic equilibrium between metarhodopsin I (meta I) and the two kinetically distinguished forms of meta II in the branched meta II model [meta IIfast and meta IIslow; Straume, M., Mitchell, D. C., Miller, J. L., & Litman, B. J. (1990) Biochemistry (preceding paper in this issue)] is derived from kinetic measurements of rhodopsin photolysis in these vesicle systems at several temperatures. Values of the meta I in equilibrium with meta IItotal equilibrium constant, Keq, are calculated from the derived model-dependent rate constants, and are shown to be equivalent to those derived from rapidly acquired absorbance spectra. The presence of 30 mol% cholesterol reduces Keq by approximately 50% between 10 and 37 degrees C. Analysis of the model-dependent parameters in terms of delta H and delta S reveals that cholesterol raises the free energy of meta IIslow, relative to meta I, by increasing delta H whereas it raises the relative free energy of meta IIfast by making delta S meta IIfast relative to meta I less positive. The reduction in Keq by both temperature and cholesterol is found to be directly correlated with a parameter that reflects the free volume available for molecular motion in the hydrophobic core of the bilayer [Straume, M., & Litman, B. J. (1988) Biochemistry 27, 7723-7733].(ABSTRACT TRUNCATED AT 250 WORDS)

SCD1 is a novel oncogenic factor specifically required for tumor cell viability in ATC. Furthermore, the expression of SCD1 appears to be correlated with thyroid tumor aggressiveness and may serve as a prognostic biomarker. These findings substantiate SCD1 as a novel tumor-specific target for therapy in patients with ATC and should be further investigated in a clinical setting.

Flash photolysis experiments designed to monitor the establishment of the metarhodopsin I to metarhodopsin II equilibrium are interpreted according to a branched model in which two spectrally indistinguishable but kinetically distinguishable forms of metarhodopsin II are postulated to exist in equilibrium with a common pool of metarhodopsin I. This interpretation arises from the consistent requirement for at least three exponentials for a valid description of the observed growth of absorbance at 380 nm following bleaching of bovine rhodopsin in rod outer segment disk membranes. Analysis of the 380-nm transient absorbance data permitted direct determination of the five physically interpretable individual rate constants of the model. This analysis represents a more explicit interpretation of kinetic data than that employed in earlier experiments of this kind, which involved estimating only apparent rates and apparent amplitudes of discrete multiexponential functions. The 380-nm absorbance contributions of all relevant species contributing to the observed dynamic absorbance change were accounted for simultaneously during nonlinear least-squares estimation of the model rate parameters. Analysis of deconvoluted equilibrium spectra acquired from samples identical with those used in the kinetics experiments confirmed the metarhodopsin I-metarhodopsin II equilibrium constants, Keq, derived from the dynamic analyses. It is shown that Keq varies from 1.28 at 10 degrees C to 7.3 at 37 degrees C and that approximately 90% of the metarhodopsin II present is in the form of metarhodopsin IIslow over the temperature range 10-37 degrees C. A physical interpretation of this decay model is discussed in the context of a distribution of metarhodopsin II structural and energetic states.

BackgroundPatient-derived xenografts (PDXs) are increasingly used in cancer research as a tool to inform cancer biology and drug response. Most available breast cancer PDXs have been generated in the metastatic setting. However, in the setting of operable breast cancer, PDX models both sensitive and resistant to chemotherapy are needed for drug development and prospective data are lacking regarding the clinical and molecular characteristics associated with PDX take rate in this setting.MethodsThe Breast Cancer Genome Guided Therapy Study (BEAUTY) is a prospective neoadjuvant chemotherapy (NAC) trial of stage I-III breast cancer patients treated with neoadjuvant weekly taxane+/-trastuzumab followed by anthracycline-based chemotherapy. Using percutaneous tumor biopsies (PTB), we established and characterized PDXs from both primary (untreated) and residual (treated) tumors. Tumor take rate was defined as percent of patients with the development of at least one stably transplantable (passed at least for four generations) xenograft that was pathologically confirmed as breast cancer.ResultsBaseline PTB samples from 113 women were implanted with an overall take rate of 27.4% (31/113). By clinical subtype, the take rate was 51.3% (20/39) in triple negative (TN) breast cancer, 26.5% (9/34) in HER2+, 5.0% (2/40) in luminal B and 0% (0/3) in luminal A. The take rate for those with pCR did not differ from those with residual disease in TN (p = 0.999) and HER2+ (p = 0.2401) tumors. The xenografts from 28 of these 31 patients were such that at least one of the xenografts generated had the same molecular subtype as the patient. Among the 35 patients with residual tumor after NAC adequate for implantation, the take rate was 17.1%. PDX response to paclitaxel mirrored the patients’ clinical response in all eight PDX tested.ConclusionsThe generation of PDX models both sensitive and resistant to standard NAC is feasible and these models exhibit similar biological and drug response characteristics as the patients’ primary tumors. Taken together, these models may be useful for biomarker discovery and future drug development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-017-0920-8) contains supplementary material, which is available to authorized users.

In the vertebrate rod outer segment (ROS), the light-dependent activation of a GTP-binding protein (G-protein) and phosphodiesterase (PDE) is quenched by a process that requires ATP [Liebman, P.A., & Pugh, E.N. (1979) Vision Res. 19, 375-380]. The ATP-dependent quenching mechanism apparently requires the phosphorylation of photoactivated rhodopsin (Rho*); however, a 48-kilodalton protein (48K protein) has also been proposed to participate in the inactivation process. Purified species of phosphorylated rhodopsin containing 0, 2, or greater than or equal to 4 (high) phosphates per rhodopsin (PO4/Rho) were reconstituted into phosphatidylcholine (PC) vesicles and reassociated with a hypotonic extract from isotonically washed disk membranes that were depleted of 48K protein; PDE activation, in response to bleaching from 0.01% to 15% of the rhodopsin present, was measured. PDE activity was reduced by at least 30% at high fractional rhodopsin bleaches and by greater than 80% at low fractional rhodopsin bleaches in high PO4/Rho samples when compared to the activity measured in O PO4/Rho controls. A phosphorylation level of 2 PO4/Rho produced PDE activities that were intermediate between O PO4/Rho and high PO4/Rho samples at low bleaches, but were identical with the O PO4/Rho samples at high rhodopsin bleaches. Rhodopsin phosphorylation is thus capable of producing a graded inhibition of light-stimulated PDE activation over a limited range of (near physiological) bleach levels. This effect become less pronounced as the bleach levels approach those that saturate PDE activation. These results are consistent with increasing levels of phosphorylation, producing a reduction of the binding affinity of G-protein for Rho*.