Transcutaneous IL-2 uptake mediated by Transfersomes depends on concentration and fractionated application

INTRODUCTION: Transfersomes (TF) are new, ultradeformable carriers with characteristics that enable them to penetrate the skin spontaneously. TFs are able to transport noninvasively both low- and high-molecular-weight molecules into the body. MATERIALS AND METHODS: TFs contain phosphatidylcholine and sodium cholate. Recombinant human interleukin-2 (Proleukin, Chiron) was added to the TFs and incubated for 24 h at 4 degrees C. The immunotransfersomes (ITF) were isolated from free interleukin-2 (IL-2) by filtration (Centrisart, Sartorius). Twenty-five thousand, 50,000 and 150,000 IU pure IL-2 and ITFs, which had been incubated with the same concentrations of IL-2, were applied subcutaneously (s.c.) (n = 8) and epicutaneously (e.c.) (n = 8) to mice. The IL-2 serum concentrations in the mice were then measured by ELISA after 2, 4, 6, 8, 10 and 24 h. Fractionation of the transdermal IL-2 application was also examined as a means of improving uptake. RESULTS: In concentrations of 25,000 and 50,000 IU IL-2, the subcutaneous application of ITFs resulted in a longer lasting IL-2 serum concentration than did the subcutaneous application of pure IL-2. While at 25,000 IU, the epicutaneous application of ITFs resulted in serum concentrations comparable to those resulting from s.c. application, at 50,000 and 150,000 IU, only 50% and 22.6% of the maximum serum concentration resulting from the s.c. application of pure IL-2 was obtained. Fractionating the transdermal IL-2 application improved uptake. CONCLUSION: We were able to show that biologically active IL-2 can be bonded to TFs up to 75%. It is possible to transport IL-2 through the skin using TFs. Both the concentration-dependent saturation of the TFs with IL-2 and fractionation of the application resulted in differing degrees of transcutaneous IL-2 uptake.     

The Limnotrons: a facility for experimental community and food web research

Experiments with multi-species communities are essential in order to get more insight in the complex interactions between organisms and their biotic and abiotic environment. The Limnotrons are aquatic model ecosystems that have been developed at the NIOO-KNAW Centre for Limnology to study pelagic community dynamics. They are suitable for the controlled study of multi-species interactions at larger spatial and temporal scales. The Limnotrons do not mimic any particular ecosystem, but should be used for the exploration of basic ecological principles. The temperature and mixing conditions in the Limnotrons can be set within narrow limits, whereas light conditions at the water surface are fixed. We show some results of system performance: mixing time, temperature control, light quantity and quality and development of a Cladocera community in a prototype of the Limnotrons. We provide results of an experiment done in four Limnotrons with the chlorophyte Scenedesmus obliquus. All trophic levels (decomposers, primary producers, and secondary producers) could be maintained in the Limnotrons for at least several weeks. Both abiotic and biotic data from the phytoplankton experiment show remarkably similar patterns through time, but had too low statistical power to prove that they are identical. We calculated the numbers of samples required for sufficient power for biomass data from two plankton experiments, and calculate required effect sizes for certain powers for a future set-up with 2×4 Limnotrons. We show that the power of the data is dependent on: the number of samples, the sample volume, the choice of the measurement method and the type of data transformation.     

Diversity of ammonifying bacteria

The diversity of the micro-organisms, involved in the ammonification in natural waters was examined. The utilization of 41 organic compounds as the sole carbon and energy source was determined for 68 isolated strains. Within this group there was hardly any specificity for the substrate. A single linkage clustering demonstrated that the bacterial population which is able to take up amino acids in natural waters, is composed of a variety of micro-organisms which only slightly differ in their ability to utilize a variety of organic compounds.     

Wide-Field Multi-Parameter FLIM: long-term minimal invasive observation of proteins in living cells

Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes.     

Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria is an obligate dimer and does not form a stable complex with the Ca(2+)-discharged photoprotein clytin

Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Fo?rster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca(2+)-discharged form that is highly fluorescent (λ(max) = 506 nm) and its GFP (cgreGFP; λ(max) = 500 nm). Ca(2+)-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca(2+)-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.     

A general approach for detecting folding intermediates from steady-state and time-resolved fluorescence of single-tryptophan-containing proteins

During denaturant-induced equilibrium (un)folding of wild-type apoflavodoxin from Azotobacter vinelandii, a molten globule-like folding intermediate is formed. This wild-type protein contains three tryptophans. In this study, we use a general approach to analyze time-resolved fluorescence and steady-state fluorescence data that are obtained upon denaturant-induced unfolding of a single-tryptophan-containing variant of apoflavodoxin [i.e., W74/F128/F167 (WFF) apoflavodoxin]. The experimental data are assembled in matrices, and subsequent singular-value decomposition of these matrices (i.e., based on either steady-state or time-resolved fluorescence data) shows the presence of three significant, and independent, components. Consequently, to further analyze the denaturation trajectories, we use a three-state protein folding model in which a folding intermediate and native and unfolded protein molecules take part. Using a global analysis procedure, we determine the relative concentrations of the species involved and show that the stability of WFF apoflavodoxin against global unfolding is ～4.1 kcal/mol. Analysis of time-resolved anisotropy data of WFF apoflavodoxin unfolding reveals the remarkable observation that W74 is equally well fixed within both the native protein and the molten globule-like folding intermediate. Slight differences between the direct environments of W74 in the folding intermediate and native protein cause different rotameric populations of the indole in both folding species as fluorescence lifetime analysis reveals. Importantly, thermodynamic analyses of the spectral denaturation trajectories of the double-tryptophan-containing protein variants WWF apoflavodoxin and WFW apoflavodoxin show that these variants are significantly more stable (5.9 kcal/mol and 6.8 kcal/mol, respectively) than WFF apoflavodoxin (4.1 kcal/mol) Hence, tryptophan residues contribute considerably to the 10.5 kcal/mol thermodynamic stability of native wild-type apoflavodoxin.     

Estimation of musculotendon kinematics in large musculoskeletal models using multidimensional B-splines

We present a robust and computationally inexpensive method to estimate the lengths and three-dimensional moment arms for a large number of musculotendon actuators of the human lower limb. Using a musculoskeletal model of the lower extremity, a set of values was established for the length of each musculotendon actuator for different lower limb generalized coordinates (joint angles). A multidimensional spline function was then used to fit these data. Muscle moment arms were obtained by differentiating the musculotendon length spline function with respect to the generalized coordinate of interest. This new method was then compared to a previously used polynomial regression method. Compared to the polynomial regression method, the multidimensional spline method produced lower errors for estimating musculotendon lengths and moment arms throughout the whole generalized coordinate workspace. The fitting accuracy was also less affected by the number of dependent degrees of freedom and by the amount of experimental data available. The spline method only required information on musculotendon lengths to estimate both musculotendon lengths and moment arms, thus relaxing data input requirements, whereas the polynomial regression requires different equations to be used for both musculotendon lengths and moment arms. Finally, we used the spline method in conjunction with an electromyography driven musculoskeletal model to estimate muscle forces under different contractile conditions, which showed that the method is suitable for the integration into large scale neuromusculoskeletal models.     

The Good,the Bad and the Plenty: Interactive Effects of Food Quality and Quantity on the Growth of Different Daphnia Species

Effects of food quality and quantity on consumers are neither independent nor interchangeable. Although consumer growth and reproduction show strong variation in relation to both food quality and quantity, the effects of food quality or food quantity have usually been studied in isolation. In two experiments, we studied the growth and reproduction in three filter-feeding freshwater zooplankton species, i.e. Daphnia galeata x hyalina, D. pulicaria and D. magna, on their algal food (Scenedesmus obliquus), varying in carbon to phosphorus (C∶P) ratios and quantities (concentrations). In the first experiment, we found a strong positive effect of the phosphorus content of food on growth of Daphnia, both in their early and late juvenile development. Variation in the relationship between the P-content of animals and their growth rate reflected interspecific differences in nutrient requirements. Although growth rates typically decreased as development neared maturation, this did not affect these species-specific couplings between growth rate and Daphnia P-content. In the second experiment, we examined the effects of food quality on Daphnia growth at different levels of food quantity. With the same decrease in P-content of food, species with higher estimated P-content at zero growth showed a larger increase in threshold food concentrations (i.e. food concentration sufficient to meet metabolic requirements but not growth). These results suggest that physiological processes such as maintenance and growth may in combination explain effects of food quality and quantity on consumers. Our study shows that differences in response to variation in food quality and quantity exist between species. As a consequence, species-specific effects of food quality on consumer growth will also determine how species deal with varying food levels, which has implications for resource-consumer interactions.     

Fluorescence of Alexa Fluor Dye Tracks Protein Folding

Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.