Conformational fluctuation of native-like and molten-globule-like cytochrome c observed by time-resolved hole burning

Nanosecond to millisecond conformational fluctuations of Zn-substituted cytochrome c (ZnCytc) have been studied by the time-resolved transient hole-burning method. The investigation of low-temperature dynamics has been made on the ZnCytc solution sample in a water-glycerol mixture. The conformational fluctuations in the native-like and the molten-globule (MG)-like states have been compared for the aqueous solution samples at room temperature. ZnCytc in the MG-like state has been prepared by adding 200 mM NaClO4 to the protein solution with a pH of 2.1, and the formation of the MG-like state has been confirmed by both the far-UV CD and the visible absorption spectra. The hole spectrum of ZnCytc has been found to consist of two nearly degenerate components, that is, the Qx and Qy bands. The temporal change in the Qx component hole spectrum has been extracted by fitting the observed hole spectrum to the three-Gaussian form. The experimental results for ZnCytc dissolved in a water-glycerol mixture have revealed that the conformational fluctuation of ZnCytc is suppressed around 200 K, which is nearly the same temperature as the glass-like transition point of Zn-substituted myoglobin (ZnMb) and also as the glass-transition point of the solvent. This supports the idea of the solvent-induced glass-like transition of a protein. It has been also found that at physiological temperatures the time scale of the conformational fluctuation of ZnCytc lies around a few tens of nanoseconds, which is 2-3 orders of magnitude faster than that of ZnMb. The experimental results for the aqueous solution samples have shown that the difference between the native-like and the MG-like states is not conspicuous. However, they are indicative of the appearance of the slower conformational fluctuation in the MG-like state.     

Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival

Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.     

Real-time observation of conformational fluctuations in Zn-substituted myoglobin by time-resolved transient hole-burning spectroscopy.

Equilibrium fluctuations of the protein conformation have been studied in myoglobin by a novel method of time-resolved transient hole-burning spectroscopy over a temperature range of 180-300 K and a time range of 10 ns to 10 ms. The temporal shift of the hole spectrum has been observed in a wide temperature region of 200-300 K. It has been found that the time behavior of the peak position of the hole is highly nonexponential and can be expressed by a stretched exponential function with a beta value of 0.22. As compared with the results for a dye solution sample, the time scale of the fluctuation of the protein conformation is much more weakly dependent on temperature. The time scale of the observed conformational dynamics shows a temperature dependence similar to that associated with the ligand escape process of myoglobin.     

Can cytological features differentiate reactive renal tubular cells from low‐grade urothelial carcinoma cells?

H. Ohsaki, E. Hirakawa, Y. Kushida, S. Yokoshita, M. Nakamura, H. Kiyomoto and R. Haba Can cytological features differentiate reactive renal tubular cells from low‐grade urothelial carcinoma cells? Objective: To compare the cytomorphological and immunocytochemical features of reactive renal tubular cells and low‐grade urothelial carcinoma cells (LG‐UCs). Methods: We examined 15 cytological parameters in 38 cases with reactive renal tubular cells in renal disease and 20 cases of LG‐UCs from bladder cancer that had been diagnosed by histological examination. Voided urine cytological parameters evaluated were as follows: (i) maximum cell numbers of clusters, (ii) cannibalism, (iii) rosette‐like arrangement, (iv) hobnail‐shaped cells, (v) vacuolated cytoplasm, (vi) intracytoplasmic haemosiderin, (vii) irregular nuclear contours, (viii) chromatin pattern, (ix) prominent nucleoli, (x) cast encasement, (xi) casts, (xii) dysmorphic erythrocytes, (xiii) isomorphic erythrocytes, (xiv) necrosis, and (xv) vimentin reactivity. The above parameters were determined using Mann–Whitney U‐test and chi‐square test, with differences considered significant at P < 0.05. Results: In reactive renal tubular cells, low to moderate cell numbers of clusters (fewer than 50 cells), rosette‐like arrangement, hobnail‐shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, euchromatin pattern, prominent nucleoli, dysmorphic erythrocytes and vimentin reactivity were present in significantly higher proportions compared with those in LG‐UCs. In LG‐UCs, high cell numbers of clusters (50 cells or more), cannibalism, heterochromatin pattern, isomorphic erythrocytes and necrosis were seen in significantly higher proportions. No significant differences were observed in irregular nuclear contours, cast encasement or casts. Conclusions: Based on results of the present study, maximum cell numbers of clusters, cannibalism, rosette‐like arrangement, hobnail‐shaped cells, vacuolated cytoplasm, intracytoplasmic haemosiderin, chromatin pattern, prominent nucleoli, dysmorphic erythrocytes, isomorphic erythrocytes, necrosis, and vimentin reactivity were capable of distinguishing reactive renal tubular cells from LG‐UCs.     

The effect of C3 transferase on human adipose-derived stem cells

Human adipose-derived stem cells (ASCs) are adult pluripotent stem cells, which have the ability to differentiate into fat, cartilage, bone, or nerves that can be applied in tissue engineering. On the other hand, the exoenzyme C3 transferase (C3) is a Rho inhibitor. Once in the cytosol, the cell-penetrating moiety is released, thereby allowing C3 transferase to freely diffuse intracellularly and inactivate RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1. In this study, we investigated ASC cytoskeletal changes induced by the addition of C3 employing immunofluorescence staining, changes in alpha-smooth muscle actin (a-SMA) gene expression employing real-time RT-PCR, and the Rho-inhibitory effect employing the pull-down assay. C3 significantly reduced stress fiber disruption and a-SMA expression 24 h after its addition at a concentration of 1 μg/ml, and it also reduced the Rho activity level. While the correlation of the occurrence can be assumed, it requires further examination to verify it. C3 may be an effective inhibitor of intracellular signal transmission in ASC cytoskeletal control involving Rho.