Reconstitution of the fusogenic activity of vesicular stomatitis virus.

Enveloped virus glycoproteins exhibit membrane fusion activity. We have analysed whether the G protein of vesicular stomatitis virus, reconstituted into liposomes, is able to fuse nucleated cells in a pH-dependent fashion. Proteoliposomes produced by octylglucoside dialysis did not exhibit cell fusion activity of the G protein. However, by making use of n-dodecyl octaethylene monoether (C12E8) as the solubilizing agent and by removal of the detergent in two steps, we were able to produce fusogenic G protein liposomes. These G protein liposomes fuse to the BHK-21 cell surface at pH 5.7-6.0 with an efficiency of fusion comparable with that of the parent virus. Physical and chemical analysis revealed that the fusogenic liposomes exhibited a protein to lipid weight ratio of 0.67 and showed an average diameter of 130 nm.     

Complement Factor H-Related Protein 3 Serum Levels Are Low Compared to Factor H and Mainly Determined by Gene Copy Number Variation in CFHR3

The major human complement regulator in blood, complement factor H (FH), has several closely related proteins, called FH-related (FHR) proteins. As all FHRs lack relevant complement regulatory activity, their physiological role is not well understood. FHR protein 3 (FHR-3) has been suggested to compete with FH for binding to Neisseria meningitidis, thereby affecting complement-mediated clearance. Clearly, the in vivo outcome of such competition greatly depends on the FH and FHR-3 concentrations. While FH levels have been established, accurate FHR-3 levels were never unequivocally reported to date. Moreover, CFHR3 gene copy numbers commonly vary, which may impact the FHR-3 concentration. Hence, we generated five anti-FHR-3 mAbs to specifically measure FHR-3 in human healthy donors of which we determined the gene copy number variation at the CFH/CFHR locus. Finally, we examined the acute-phase response characteristics of FHR-3 in a small sepsis cohort. We determined FHR-3 levels to have a mean of 19 nM and that under normal conditions the copy number of CFHR3 correlates to a very large extent with the FHR-3 serum levels. On average, FHR-3 was 132-fold lower compared to the FH concentration in the same serum samples and FHR-3 did not behave as a major acute phase response protein.     

A new approach to polarized fluorescence using phase and modulation fluorometry

In the preceding paper, an alternative method is described for obtaining information about the reorientational behavior of a fluorophore in a membrane system from frequency domain measurements. To demonstrate this new analysis procedure, we present data for the probe-molecule 1,6-diphenyl-1,3,5-hexatriene (DPH) in l--dimyristoyl- and l--dipalmitoylphosphatidylcholine (DMPC and DPPC) obtained with two different phase fluorometers: the SLM 4800A Subnanosecond Spectrofluorometer which has only three fixed frequencies available (6, 18 and 30 MHz) and the recently constructed continuously variable multifrequency phasefluorometer (Gratton and Limkeman 1983).It will be shown that reasonable information about the anisotropy behavior of a fluorophore can be obtained even if only three frequencies are used. The phase modulation technique was also used to check the new expression for the anisotropy, r(t), called the general model and introduced by Van der Meer et al. (1984). The parameters P ₂, P ₄ and D, obtained from the nonlinear least squares fit (Bevington 1969) for this general model, confirm the results from the pulse technique of Ameloot and coworkers (Ameloot et al. 1984; Pottel et al. 1986).     

Transformation of Brassica napus and Brassica oleracea Using Agrobacterium tumefaciens and the Expression of the bar and neo Genes in the Transgenic Plants

An efficient and largely genotype-independent transformation method for Brassica napus and Brassica oleracea was established based on neo or bar as selectable marker genes. Hypocotyl explants of Brassica napus and Brassica oleracea cultivars were infected with Agrobacterium strains containing chimeric neo and bar genes. The use of AgNO₃ was a prerequisite for efficient shoot regeneration under selective conditions. Vitrification was avoided by decreasing the water potential of the medium, by decreasing the relative humidity in the tissue culture vessel, and by lowering the cytokinin concentration. In this way, rooted transformed shoots were obtained with a 30% efficiency in 9 to 12 weeks. Southern blottings and genetic analysis of S1-progeny showed that the transformants contained on average between one and three copies of the chimeric genes. A wide range of expression levels of the chimeric genes was observed among independent transformants. Up to 25% of the transformants showed no detectable phosphinotricin acetyltransferase or neomycin phosphotransferase II enzyme activities although Southern blottings demonstrated that these plants were indeed transformed.     

Reclustering of scattered Golgi elements occurs along microtubules

Depolymerization of the interphase microtubules by nocodazole results in the scattering and apparent fragmentation of the Golgi apparatus in Vero fibroblast cells. Upon removal of the drug, the interphase microtubules repolymerize, and the scattered Golgi elements move back to the region around the microtubule-organizing center (MTOC) within 40 to 60 min. Using a fluorescent lipid analogue (C6-NBD-ceramide) as a vital stain for the scattered Golgi elements, their relocation was visualized by video-enhanced fluorescence microscopy in Vero cells maintained at 20 degrees C. The NBD-labeled structures were identified as Golgi elements by their colocalization with galactosyltransferase in the fixed cells. During reclustering, NBD-labeled Golgi elements were observed to move by discontinuous saltations towards the MTOC with velocities of 0.1 to 0.4 micron/s. Paths along which Golgi elements moved were super-imposable on microtubules visualized by indirect immunofluorescence. Neither the collapse of intermediate filaments caused by microinjection of antibodies to vimentin nor the disruption of microfilaments by cytochalasin D had an effect on the reclustering of Golgi elements or the positioning of the Golgi apparatus. These data show that scattered Golgi elements move along microtubules back to the region around the MTOC, while neither intact intermediate filaments nor microfilaments are involved.     

Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids.

Transbilayer migration of membrane phospholipid arising from membrane insertion of the terminal human complement proteins has been investigated. Asymmetric vesicles containing pyrene-labeled phosphatidylcholine (pyrenePC) concentrated in the inner monolayer were prepared by outer monolayer exchange between pyrenePC-containing large unilamellar vesicles and excess (unlabeled) small unilamellar vesicles, using bovine liver phosphatidylcholine-specific exchange protein. After depletion of pyrenePC from the outer monolayer, the asymmetric large unilamellar vesicles were isolated by gel filtration and exposed to the purified C5b-9 proteins at 37 degrees C. Transbilayer exchange of phospholipid between inner and outer monolayers during C5b-9 assembly was monitored by changes in pyrene excimer and monomer fluorescence. Membrane deposition of the C5b67 complex (by incubation with C5b6 + C7) caused no change in pyrenePC fluorescence. Addition of C8 to the C5b67 vesicles resulted in a dose-dependent decrease in the excimer/monomer ratio. This change was observed both in the presence and absence of complement C9. No change in fluorescence was observed for control vesicles exposed to C8 (in the absence of membrane C5b67), or upon C5b-9 addition to vesicles containing pyrenePC symmetrically distributed between inner and outer monolayers. These data suggest that a transbilayer exchange of phospholipid between inner and outer monolayers is initiated upon C8 binding to C5b67. The fluorescence data were analyzed according to a "random walk" model for excimer formation developed for the case where pyrenePC is asymmetrically distributed between lipid bilayers. Based on this analysis, we estimate that a net transbilayer migration of approximately 1% of total membrane phospholipid is initiated upon C8 binding to C5b67. The potential significance of this transbilayer exchange of membrane phospholipid to the biological activity of the terminal complement proteins is considered.