The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

We have studied oscillating glycolysis in the strain BY4743 and isogenic strains with deletions of genes encoding enzymes in glycolysis, mitochondrial electron transport and ATP synthesis. We found that deletion of the gene encoding the hexokinase 1 isoform does not affect the oscillations while deletion of the gene encoding the hexokinase 2 isoform results in oscillations with smaller amplitude. The latter is associated with an almost 50% decrease in hexokinase activity. Deletions in the genes encoding the a-and b-subunits of phosphofructokinase abolish the oscillations entirely. This loss in oscillatory activity is associated with a fourfold decrease in phosphofructokinase activity. Deletions of genes encoding subunits of the F 1 F 0 ATPase also inhibit the oscillations in accordance with earlier studies using for example inhibitors. Finally, we identified an apparently new control point involving the mitochondrial cytochrome c oxidase. The latter is difficult to explain as oscillatory activity entails 100% inhibition of this enzyme. The mitochondria of this strain seem to have normal F 1 F 0 ATPase activity. Overall these results support earlier experimental and model studies suggesting that in addition to processes within glycolysis also processes outside this pathway contribute to the control of the oscillatory behaviour.

Arenicola Marina extracellular hemoglobin (Hbl Hb) is considered to be a promising candidate as a blood substitute. To entangle some of the properties of extracellular giant hexagonal bilayer hemoglobin (Hbl Hb) of Arenicola Marina, we combined polarized resonance Raman scattering (532 nm excitation) with dynamic light scattering (DLS) (632.8 nm). An analysis of the depolarization ratio of selected a(2g) skeletal modes of the heme in native Hbl Hb and porcine Hb, shows that the distortion of the heme group away from its ideal fourfold symmetry is much smaller for heme groups bound in the Hbl Hb than for heme groups bound in porcine Hb. Using DLS, the average hydrodynamic diameter () of Hbl Hb was measured at pH = 5, 7, 8, 9, and 10. At pH = 5 to 7, the Hbl Hb was found in its native form with equal to 24.2 nm, while at pH = 8 and 9, a dissociation process starts to take place resulting in = 9 nm. At pH = 10, only large aggregates of fragmented Hbl Hb with larger than 1000 nm was detected, however, a comparison of the DLS results with the polarized resonance Raman scattering (RRS) revealed that the coupling between the fragments did not involve direct interaction between the heme groups, but also that the local heme environment seems to be comparable in the aggregates and in the native Hbl Hb. By comparing the unpolarized RRS results obtained for erythrocytes (RBC) with those for Hbl Hb, led us to the important conclusion that Hbl Hb is much easier photolyzed than porcine RBC.

The polarization properties of surface enhanced resonance Raman scattering (SE(R)RS) of rhodamine 6G molecules, adsorbed to a hexagonally ordered gold nanostructure, are studied with the purpose to discriminate between adsorption sites with different plasmonic properties. The nanostructure is based on a self-organizing hexagonally ordered porous Al(2)O(3) substrate sputter-coated with gold. Each hexagonal subunit has D(6h) symmetry, where the symmetry center may act as an isotropic site, whereas the six narrow gaps between the individual Au hemispheres may act as hot-spots. The variation of the depolarization ratio (DPR), measured in resonance for the eight most prominent vibrational modes of the xanthene moiety, is analyzed by rotating the sample. According to theory, the DPR of the SE(R)RS signal obtained from molecules physisorbed in the isotropic sites deviates from the DPR originating from molecules physisorbed in the hot-spots in two ways: 1. The DPR associated with the isotropic sites depends differently on the rotation angle than the DPR associated with the hot-spots. 2. The DPR of the SE(R)RS signal obtained from molecules physisorbed in the isotropic sites depends on the nature of the Raman modes, whereas it for molecules physisorbed in the hot-spots is independent of the nature of the Raman modes. By applying the latter in the analysis of the polarized SE(R)RS data, we conclude that the dominating SE(R)RS signal comes from molecules adsorbed in the hot-spots. However, since the DPR's obtained for Raman modes of different symmetry are slightly different, the SE(R)RS signal must contain an additional contribution. Our analysis shows that the small mode-dependent SE(R)RS signal most likely comes from molecules adsorbed in the isotropic sites. The general result that can be derived from the present study is that by measuring the polarization properties in SE(R)RS and SERS it is possible to discriminate between adsorption sites with different plasmonic properties present in a highly symmetric nanostructure, even when the magnitude of the different contributions are highly different. The consequence of the insufficient spatial resolution with respect to a detailed mapping of the substrate often encountered in unpolarized SE(R)RS and in two-photon luminescence microscopy may thereby be circumvented.

The aim of the present paper is to analyze reflectance and transmittance measurements on small scattering and absorbing samples. The long term goal is to perform quantitative, spectroscopic in vivo measurements of pigments in small samples of plant material. Small samples such as small leaves constitute a special experimental challenge in cases in which the sample beam has a larger cross-sectional area than the sample. The experimental errors introduced when measuring reflectance and transmittance on small absorbing and scattering samples are investigated theoretically and experimentally by using a blue polyester sample as an appropriate test sample. The experiments are performed with either a mask or a lens setup combined with a mask inserted in the sample beam. In particular, the errors introduced in the reflectance measurements can be very large and larger than 100%. It is shown that any direct illumination of the mask must be avoided. To obtain more accurate values for the reflection coefficient it is necessary to combine the mask with a focusing lens system, adjust the mask and sample very carefully, and choose the ratio between the aperture of the mask and the beam area as large as possible. In the case of transmittance measurements, it is shown that the combination of a special sample fixture and a lens system gives rise to smaller errors compared to the errors introduced by the mask alone or the mask combined with a focusing lens system.