Aluminium foil as a single-use substrate for MALDI-MS fingerprinting of different melanoma cell lines.

Herein, we present the intact cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the fingerprinting of human melanoma cancer cell lines grown on aluminium foil. To perform the MALDI-MS assay, melanoma cells were cultured on a flat and thin foil, which was directly transferred to the target plate of MALDI-MS for analysis. The influence of a wide range of cell fixation protocols (i.e. formalin-based and alcohol-based methods) and MALDI matrices on the obtained characteristic spectra was investigated. For the optimization of the MALDI-MS protocol, the MS fingerprints of the melanoma WM-239 cell line with and without an overexpressed enhanced green fluorescent protein were employed. The fingerprints obtained from WM-239 cells grown on aluminium foil were compared with the intact cell MALDI-MS of the cell pellet and presented higher sensitivity in a high m/z range. The optimized protocol was subsequently applied to characterise melanoma cell lines derived from different cancer stages and allowed identification of unique MS signals that could be used for differentiation between the studied cell lines (i.e. molecular weight equal to 10.0 kDa and 26.1 kDa).

Visualizing odorant receptor trafficking in living cells down to the single-molecule level.

Despite the importance of trafficking for regulating G protein-coupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulse-labeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Single-molecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of approximately 190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception.

Balancing GFP reporter plasmid quantity in large-scale transient transfections for recombinant anti-human Rhesus-D IgG1 synthesis.

Using transient expression, high amounts (>20 mg/mL) of secreted anti-human Rhesus-D IgG1 were produced in a suspension-adapted HEK293 EBNA cell line (Meissner et al., Biotechnol Bioeng 75: 197-203, 2001). Time of harvest was 3 days after transfection. For the estimation of transfection efficiencies, we routinely co-transfected EGFP reporter DNA. At higher reporter plasmid concentrations, >2% of total transfecting plasmid DNA, a substantial reduction of recombinant antibody synthesis, was observed. This phenomenon was investigated in detail by co-expressing various green fluorescent protein (GFP) reporter constructs, which were targeted at different subcellular locations. Enhanced and humanized GFPs targeted to either the endoplasmic reticulum, the cytosol, or the nucleus reduced recombinant antibody production by 30 to 40% when present at higher concentrations in the transfection solution. The most severe effects were observed when the co-transfected EGFP was targeted to the endoplasmic reticulum, leading to a reduction of up to 80% in the presence of only 5% of reporter DNA. Interestingly, one nuclear-targeted GFP variant that was not codon optimized for expression in human cell lines could be added, to up to almost half of the total amount of transfecting DNA, without adverse effect on antibody production. Although the minimum amount of this reporter DNA needed for fluorescence reading was 10 times higher than for the other variants, it provided a much broader quantity range within which the transfection process could be studied without being negatively affected.

Mapping the antagonist binding site of the serotonin type 3 receptor by fluorescence resonance energy transfer.

We have measured fluorescence resonance energy transfer (FRET) between a fluorescent antagonist, bound to the purified detergent-solubilized serotonin type 3 receptor, and a lipophilic acceptor probe partitioned into the micelle surrounding the detergent-solubilized receptor. The experimentally observed FRET efficiency was evaluated on the basis of the characteristic dimensions of the receptor-micelle complex and the average number of acceptor molecules in such micelles. The binding site was determined to be 5.4 +/- 0.9 nm above the center of the detergent micelle. The experiments were performed below the critical micellar concentration of the detergent (C(12)E(9)) used to solubilize the receptor, under which conditions it was demonstrated that the ligand binding activity was fully preserved. This reduces considerably the fluorescence background arising from probes not associated with the receptor, allowing a precise determination of the transfer efficiency.

Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells.

Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.

In vitro and in vivo ligand binding to the 5HT(3) serotonin receptor characterised by time-resolved fluorescence spectroscopy.

The binding of the fluorescein-labelled antagonist GR-flu ([1,2,3,9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluoresceinthiocarbamoyl)propyl)-4H-carbazol-4-one]) to a purified, detergent-solubilised ligand-gated ion channel, the type-3 serotonin (5-hydroxytryptamine, 5HT) receptor (5HT(3)R), was characterised by frequency-domain time-resolved fluorescence spectroscopy (TRFS). Detailed understanding of how ligands interact with the homopentameric receptor was obtained. While a 1:1 stoichiometry was observed for the GR-flu-receptor complex, the agonist quipazine bound cooperatively to the receptor, suggesting multiple binding sites for this ligand. The GR-flu-binding site of the receptor was proven to provide an acidic environment as shown by determining the fraction of bound GR-flu in the protonated state. Fluorescence anisotropy relaxation experiments indicated a hindered but still high mobility for the receptor-bound GR-flu. Hence, the binding site is expected to present a wide opening to the ligand. Finally, we succeeded in measuring the binding of GR-flu to 5HT(3) receptors in live cells. These results show that the purified and the native receptor behave identically and demonstrate that time-resolved fluorescence measurements are suited to selectively investigate biomolecular interactions in live cells.

Integrating basic and applied developmental research: a new model for the twenty-first century.

Until recently, basic and applied research agendas in the field of child development have followed separate paths. One reason the two have not merged is that the objectives of basic and applied research are often seen as incompatible. In this paper, we argue that researchers can simultaneously achieve the objectives of advancing basic knowledge and addressing applied problems within a single research program. We provide a framework for this perspective by first looking back at historical trends of basic and applied developmental research and then looking forward at potential new approaches for integrating basic and applied research. We use our own research on perception of affordances and unintentional childhood injuries to illustrate how researchers might implement these strategies for integrating basic and applied research. We conclude by discussing how we might extend this integration further to include nontraditional classes of application.

Ligand binding to the serotonin 5HT3 receptor studied with a novel fluorescent ligand.

The thermodynamics and kinetics of ligand binding to the purified serotonin 5HT3 receptor and the local environment of the bound ligand were studied by fluorescence spectroscopy using a novel fluorescein-labeled ligand GR-flu [1,2,3, 9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-amino-(N-fluo rescien-thiocarbamoyl)-propyl)-4H-carbazol-4-one]. Electrophysiological investigations demonstrated GR-flu to be an antagonist, and radioligand competition assays delivered a dissociation constant of 0.32 nM. Changes in the fluorescence intensity and anisotropy upon specific binding to the receptor yielded dissociation constants of approximately 0.2 nM. Fluorescence measurements showed that selective 5HT3 receptor ligands competed for GR-flu binding with a rank order of potency identical to that established with the radioligand [3H]-GR65630. The kinetics of GR-flu binding to the 5HT3 receptor revealed a bimolecular association process with an on-rate constant of 1.17 x 10(6) s-1 M-1 and a biphasic dissociation reaction with off-rate constants of 275 x 10(-)6 and 43 x 10(-)6 s-1. The temperature dependence of the dissociation constant yielded an enthalpic term of -26 kJ mol-1 and an entropic term of 94 J K-1 mol-1 for the binding of GR-flu to the receptor, indicating that both quantities contribute equally to the reaction. An activation enthalpy DeltaH#on and entropy DeltaS#on of binding of 50 kJ mol-1 and 43 J mol-1 K-1 were obtained, indicating that the entropy facilitates the initial steps of GR-flu binding to the 5HT3 receptor. The fluorescence anisotropy of receptor-bound GR-flu and the environmental sensitivity of the fluorescent probe suggest that the binding site has a wide entrance and that it is 0.8 pH unit more acidic than the bulk solution.

Effects of distance on vocal intensity.

The vocal response of speakers to change of distance from a listener is in dispute. Warren (1968) found that speakers obeyed the inverse square law when compensating for distance changes; that is, they decreased their vocal intensity by 6 dB when distance was halved. However, speakers in a study of Johnson, Pick, Siegel, Cicciarelli, and Garber (1981) changed their vocal intensity by much less than 6 dB. This study was an attempt to reconcile the conflicting results and to gain better understanding of what people know implicitly about the effects of distance on intensity. Speakers in the present study significantly changed their vocal intensity to compensate for changes in distance, but by a maximum of 2.46 dB. Possible reasons for the different results are discussed.

Calibration of human locomotion and models of perceptual-motor organization.

People coordinate the force and direction of skilled actions with target locations and adjust the calibrations to compensate for changing circumstances. Are the adjustments globally organized (adjusting a particular action to fit a particular circumstance would generalize to all actions in the same circumstance); anatomically specific (every effector is adjusted independently of others); of functional (adjustments would generalize to all actions serving the same goal and generating the same perceptible consequences)? Across 10 experiments, changes in the calibration of walking, throwing, and turning-in-place were induced, and generalization of changes in calibration to functionally related and unrelated actions were tested. The experiments demonstrate that humans rapidly adjust the calibration of their walking, turning, and throwing to changing circumstances, and a functional model of perceptual-motor organization is suggested.

The yeast centromere CDEI/Cpf1 complex: differences between in vitro binding and in vivo function.

The centromere and promoter factor Cpf1 binds centromere DNA element I found in all centromere DNAs from the yeast Saccharomyces cerevisiae. We analyzed thirty different point mutations in or around CEN6-CDEI (ATCACGTG) for their relative binding affinity to Cpf1 and these data were compared with the in vivo centromere function of these mutants. We show that the minimal length of the Cpf1 binding site needed for full in vitro binding and in vivo activity is 10 base pairs long comprised of CDEI plus the two base pairs 3' of this sequence. The palindromic core sequence CACGTG is most important for in vivo CEN function and in vitro Cpf1 binding. Symmetrical mutations in either halfsite of the core sequence affect in vitro Cpf1 binding and in vivo mitotic centromere function asymmetrically albeit to a different extent. Enlarging the CDEI palindrome to 12 or 20 bps increases in vitro Cpf1 binding but results in increased chromosome loss rates suggesting a need for asymmetrical Cpf1 binding sequences. Additionally, the ability of Cpf1 protein to bind a mutant CDEI element in vitro does not parallel the ability of that mutant to confer in vivo CEN activity. Our data indicate that the in vitro binding characteristics of Cpf1 to CDEI only partly overlap with their corresponding activity within the centromere complex, thus suggesting that in the in vivo situation the CDEI/Cpf1 complex might undergo interactions with other centromere DNA/protein complexes.

The role of binocular information in ball catching.

The role of binocular vision in a ball-catching task involving spatial uncertainty was examined in three experiments. In all three experiments, subjects' catching performance was evaluated during monocular and binocular viewing, in normal room lighting and in complete darkness with a luminescent ball. Subjects' performance was found to be significantly better with binocular than with monocular vision, especially under normal lighting conditions. In the second and third experiments, catching performance was evaluated in the presence of minimal visual frames, consisting of a series of light-emitting diodes (LEDs). In Experiment 2, the visual frame consisted of a single plane of LEDs, whereas in Experiment 3, the visual frame consisted of two planes of LEDs. Catching performance was found to be significantly better with the visual frame than in complete darkness, but this was true only for binocular viewing. This result supports the hypothesis that binocular convergence is used to scale perceived space and that this information enables subjects to contact the ball successfully. It was further found that postural sway varied between lighting conditions and that less sway was accompanied by higher performance. There was no effect of binocular viewing in this respect. In general, the results suggest two additive effects of viewing conditions: a direct effect of binocular vision on ball catching and an indirect effect of lighting on postural stability, which, in turn, affects catching performance.

The independence of horizontal and vertical dimensions in handwriting with and without vision.

The purpose of this study was to investigate the relationship between horizontal and vertical components of handwriting production when subjects were instructed to vary the size of these components separately or together. The effect of vision on these instructed size transformations also was examined. Eight female adults participated in the experiment. The basic task was to write the words 'poppy' and 'wood' cursively five times, the first time in their normal size and then with four size transformations. These transformations--one-fourth, one-half, double, and four-times their normal size--were made under three different sets of instructions (width only, height only, both) and in two visual conditions (normal, blindfolded), for a total of six sets of five repetitions. The individual slopes (changes in actual values across the transformation values) for width and height under instructions to change both parameters were almost identical to the width and height slopes under instructions to change only the single parameter, supporting the notion of the independence of the horizontal and vertical components. Further, an analysis of these individual slopes indicated that the size transformations were significantly greater (p less than 0.05) (and closer to the instructed values) with vision than without vision.

Children's use of frames of reference in communication of spatial location.

The frames of reference used by 4-, 6-, and 8-year-old children were studied in a spatial direction-giving task. Children were asked to specify verbally the location of a toy hidden under one of several identical cups. The child and listener sat facing each other at opposite ends of a room that had distinctive or nondistinctive landmarks proximal and distal to the hiding location. Location needed to be specified with respect to either the left-right dimension, the front-back dimension, or both. The results indicated that (1) although children's overall performance improved with age, communication about the left-right dimension was particularly difficult for 4-year-old children and showed a higher rate of improvement with age than communication about the front-back dimension; and (2) the frames of reference that children incorporated into their directions changed with age and differed for directions about the front-back and left-right dimensions. Both 4- and 6-year-old children used person references (themselves or the listener) to specify front-back relations, but only the 6-year-olds were able to compensate for their apparent difficulty in using the terms left and right by using landmarks to specify the left-right dimension. Eight-year-olds used a combination of person and landmark references in directions about both dimensions. Discrepancies between the frames of reference children used to communicate spatial location and those typically used in other spatial cognition tasks are discussed in terms of developmental and task constraints.

Inhibiting the Lombard effect.

The Lombard effect is the tendency to increase one's vocal intensity in noise. The present study reports three experiments that test the robustness of the Lombard effect when speakers are given instructions and training with visual feedback to help suppress it. The Lombard effect was found to be extremely stable and robust. Instructions alone had little influence on the response to the noise among untrained speakers. When visual feedback correlated with vocal intensity was presented, however, subjects could inhibit the Lombard response. Furthermore, the inhibition remained after the visual feedback was removed. The data are interpreted as indicating that the Lombard response is largely automatic and not ordinarily under volitional control. When subjects do learn to suppress the effect, they seem to do so by changing overall vocal level rather than their specific response to the noise.

Role of visual information in ball catching.

The present study is concerned with the perceptual information about the body and space underlying the act of catching a ball. In a series of four experiments, subjects were asked to catch a luminous ball under various visual conditions. In general, catching in a normally illuminated room was contrasted with catching the luminous ball in an otherwise completely dark room. In the third and fourth experiments, intermediate conditions of visual information were included. The results suggest that it is possible to catch a ball with one hand when only the ball is visible, but performance is better when the subject has the benefit of a rich visual environment and two hands. The second experiment indicated that subject performance does improve with practice in the dark, but time spent in the darkened room itself doesn't result in a significant decrement in performance. Results of the third study suggest that vision of one's hand does not aid in the performance of this task whereas the presence of a minimal visual frame appears to aid performance. The final study examined the relation between catching performance and body sway under similar visual conditions. Results of this experiment imply that persons who exhibit relatively little postural sway in full-room lighting performed better at this catching task.