Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy

The green fluorescent protein (GFP) has proven to be an excellent fluorescent marker for protein expression and localisation in living cells [1] [2] [3] [4] [5]. Several mutant GFPs with distinct fluorescence excitation and emission spectra have been engineered for intended use in multi-labelling experiments [6] [7] [8] [9]. Discrimination of these co-expressed GFP variants by wavelength is hampered, however, by a high degree of spectral overlap, low quantum efficiencies and extinction coefficients [10], or rapid photobleaching [6]. Using fluorescence lifetime imaging microscopy (FLIM) [11] [12] [13] [14] [15] [16], four GFP variants were shown to have distinguishable fluorescence lifetimes. Among these was a new variant (YFP5) with spectral characteristics reminiscent of yellow fluorescent protein [8] and a comparatively long fluorescence lifetime. The fluorescence intensities of co-expressed spectrally similar GFP variants (either alone or as fusion proteins) were separated using lifetime images obtained with FLIM at a single excitation wavelength and using a single broad band emission filter. Fluorescence lifetime imaging opens up an additional spectroscopic dimension to wavelength through which novel GFP variants can be selected to extend the number of protein processes that can be imaged simultaneously in cells.     

Solar lentigines are strongly related to sun exposure in contrast to ephelides

Solar lentigines and ephelides are different types of pigmented skin lesions predominantly present on sun-exposed skin. Both lesions are risk indicators for melanoma and non-melanoma skin cancer. Solar lentigines are considered as a sign of photodamage although well-conducted epidemiological studies are lacking on this subject. Ephelides are associated with fair skin type and red hair. The aim of the present study was to investigate the relation of sun-exposure estimates with solar lentigines and ephelides. In the Leiden Skin cancer Study 577 patients with malignant melanoma and/or non-melanoma skin cancer and 385 individuals without a history of skin cancer were studied. The presence of solar lentigines and ephelides in the face and on the back was assessed. Data on skin type, hair color, sun-exposure variables and cutaneous signs of photodamage were collected, by questionnaire and physical examination. Data were analyzed by chi-square or Student t-tests and with multivariable regression. Exposure odds ratios with 95% confidence intervals (95% CI) were calculated to estimate the relative risk for the presence of solar lentigines and ephelides dependent on signs of photodamage. The association with age was strongly positive for solar lentigines whereas it was strongly negative for ephelides (P-values for trend <0.0001). After adjustment for age, sex and skin type, solar lentigines on the back were positively associated with cumulative (P = 0.01) and intermittent (P = 0.0002) sun exposure. After adjustment, solar lentigines on the back were also associated with a history of sunburns before the age of 20 yr (P = 0.0003) and the number of sunburns in childhood (P = 0.002). Solar lentigines in the face were significantly associated with cutaneous signs of photodamage, i.e. elastosis (odds ratio 2.4, 95% CI 1.7-3.3) and actinic keratosis (odds ratio 1.8, 95% CI 1.3-2.4) whereas ephelides were not. Ephelides in the face and on the back showed an inverse association with chronic sun exposure but after adjustment theses associations disappeared. Sunburns before the age of 20 appeared to be positively associated with ephelides on the back (P = 0.04). In contrast to lentigines, ephelides were much more associated with constitutional host factors such as fair skin and/or red hair (both P < 0.0001). This study indicates that both chronic and acute sun exposure are important in the pathogenesis of solar lentigines.     

Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate

The mitogen-activated protein kinase (MAPK) network is a conserved signalling module that regulates cell fate by transducing a myriad of growth-factor signals. The ability of this network to coordinate and process a variety of inputs from different growth-factor receptors into specific biological responses is, however, still not understood. We investigated how the MAPK network brings about signal specificity in PC-12 cells, a model for neuronal differentiation. Reverse engineering by modular-response analysis uncovered topological differences in the MAPK core network dependent on whether cells were activated with epidermal or neuronal growth factor (EGF or NGF). On EGF stimulation, the network exhibited negative feedback only, whereas a positive feedback was apparent on NGF stimulation. The latter allows for bi-stable Erk activation dynamics, which were indeed observed. By rewiring these regulatory feedbacks, we were able to reverse the specific cell responses to EGF and NGF. These results show that growth factor context determines the topology of the MAPK signalling network and that the resulting dynamics govern cell fate.     

Ultrastructural localization of amiloride-sensitive sodium channels and Na+,K(+)-ATPase in the rat's olfactory epithelial surface

Several studies have indicated that olfactory responses are impeded byamiloride. Therefore, it was of interest to see whether, and if so which,olfactory epithelial cellular compartments have amiloride- sensitivestructures. Using ultrastructural methods that involved rapid freezing,freeze-substitution and low temperature embedding of olfactory epithelia,this study shows that, in the rat, this tissue is immunoreactive toantibodies against amiloride sensitive Na(+)- channels. However, microvilliof olfactory supporting cells, as opposed to receptor cilia, contained mostof the immunoreactive sites. Apices from which the microvilli sprout andreceptor cell dendritic knobs had much less if any of theamiloride-antibody binding sites. Using a direct ligand-bindingcytochemical method, this study also confirms earlier ones that showed thatolfactory receptor cell cilia have Na+, K(+)-ATPase. It is proposed thatsupporting cell microvilli and the receptor cilia themselves havemechanisms, different but likely complementary, that participate inregulating the salt concentration around the receptor cell cilia. In thisway, both structures help to provide the ambient mucous environment forreceptor cells to function properly. This regulation of the saltconcentration of an ambient fluid environment is a function that theolfactory epithelium shares with cells of transporting epithelia, such asthose of kidney.     

The GDI-like solubilizing factor PDEδ sustains the spatial organization and signalling of Ras family proteins

We identify a role for the GDI-like solubilizing factor (GSF) PDEδ in modulating signalling through Ras family G proteins by sustaining their dynamic distribution in cellular membranes. We show that the GDI-like pocket of PDEδ binds and solubilizes farnesylated Ras proteins, thereby enhancing their diffusion in the cytoplasm. This mechanism allows more effective trapping of depalmitoylated Ras proteins at the Golgi and polycationic Ras proteins at the plasma membrane to counter the entropic tendency to distribute these proteins over all intracellular membranes. Thus, PDEδ activity augments K/Hras signalling by enriching Ras at the plasma membrane; conversely, PDEδ down-modulation randomizes Ras distributions to all membranes in the cell and suppresses regulated signalling through wild-type Ras and also constitutive oncogenic Ras signalling in cancer cells. Our findings link the activity of PDEδ in determining Ras protein topography to Ras-dependent signalling.     

PCR反应混合液的冻干处理及其应用

本研究将除模板DNA以外的PCR反应混合液,经过真空浓缩冻成干粉状后,分别置于室温和4℃贮存,经过一定的贮藏时间后进行PCR反应,发现经冻干处理的样品能在较长的贮存时间内保持扩增活性,这将给实际工作带来很大的便利。Abstract:In this paper we introduce a new storage method of PCR ingredient.PCR mixture except DNA template has been frozen to dry powder by the DNA-Plus system.This kind of powder was stored at room temperature or 4℃.PCR has been run in different period of storage.It was discovered that the samples of lyophilization could keep activity for a long time.