Marc Schneider

Human PRP4 kinase is required for stable tri-snRNP association during spliceosomal B complex formation

Reversible protein phosphorylation has an essential role during pre-mRNA splicing. Here we identify two previously unidentified phosphoproteins in the human spliceosomal B complex, namely the pre-mRNA processing factors PRP6 and PRP31, both components of the U4/U6−U5 tri-small nuclear ribonucleoprotein (snRNP). We provide evidence that PRP6 and PRP31 are directly phosphorylated by human PRP4 kinase (PRP4K) concomitant with their incorporation into B complexes. Immunodepletion and complementation studies with HeLa splicing extracts revealed that active human PRP4K is required for the phosphorylation of PRP6 and PRP31 and for the assembly of stable, functional B complexes. Thus, the phosphorylation of PRP6 and PRP31 is likely to have a key role during spliceosome assembly. Our data provide new insights into the molecular mechanism by which PRP4K contributes to splicing. They further indicate that numerous phosphorylation events contribute to spliceosome assembly and, thus, that splicing can potentially be modulated at multiple regulatory checkpoints.

Template-Assisted Polyelectrolyte Encapsulation of Nanoparticles into Dispersible, Hierarchically Nanostructured Microfibers

Nanoporous membranes are used as templates for polyelectrolyte encapsulation of colloidal nanoparticles, leading to colloidally dispersible, multifunctional, and multicompartment microfibers. The protocol is simple and independent of the chemical nature of the nanoparticles and polyelectrolytes. Furthermore, the surface polyelectrolyte coating allows free transfer of the resulting microfibers into various aqueous and organic media. Copyright

Structure of transfection-active histone H1/DNA complexes

Relationships between the structure of transfecting complexes of histone H1 and DNA and their transfection efficiency were studied. Transfection activity proved to be connected to complex aggregates. Low speed centrifugation of the complexes resulted in loss of the transfection activity. The complexes/aggregates were active with high efficiency in a broad range of weight input ratios ri (0.1<ri<30). Using atomic force microscopy (AFM), the complexes were imaged at negative, nearly electroneutral and positive charge conditions. Electroneutral complexes at ri=1 showed a multitude of different complex forms. Fibrillar, network-like and branched structures were frequently present in one complex. Strongly positive charged complexes had a toroidal appearance. All these different forms contributed to the high transfection efficiency. Cellular uptake is supposed to be by phagocytosis.

Characterization of structure and mechanism of transfection-active peptide-DNA complexes

We studied a number of physicochemical parameters of transfection-active peptide-DNA complexes including size, aggregation behaviour and circular dichroism (CD) spectra. These data were brought in relationship to the transfection activity of these peptides in order to better understand the mechanism of peptide-mediated gene transfer. A DNA binding oligolysine (K(16)) and a peptide comprising K(16) with an added peptide loop containing the arbitrary sequence RAD not known as a receptor ligand were used. Whereas the K(16)-DNA complex at 88% charge neutralization of the DNA phosphates collapsed into small toroidal particles with a diameter of 200 nm by dynamic light scattering, K(16)-cRAD did not. Instead, large aggregates were observed. CD spectra showed that the K(16)-DNA complexes were in a -psi state observed at liquid crystalline phases. Increasing positive charge by addition of further K(16) or disturbing the -psi state by introducing the RAD-peptide loop resulted in increasing instability indicated by aggregation and loss of the -psi CD spectrum of the complexes. Transfection experiments indicated that the aggregated material was the transfection-active component.

Quantitative measurement of chromium's ability to promote adhesion

Using atomic force spectroscopy, we investigated the adhesion-promoting ability of chromium. An intermediate layer of chromium can overcome the low adhesion between metal films and silicon dioxide. For the first time, we quantitatively studied this experimentally well known fact. We compared the adhesion between chromium and different substrates such as gold, silver, mica, and silicon dioxide and, beyond that, the adhesion between silicon dioxide and the same substrates. To avoid additional effects due to water, we chose ethanol as a nonpolar solvent. Taking the interfacial energies of the surfaces with the liquid into account eliminates the direct influence of the fluid medium on the adhesion of the solid material. The results we obtained corroborate the experimental fact of higher adhesion of chromium with the chosen substrates, as well as substantiate the value of chromium as an adhesion promoter. The adhesion of chromium-coated probes on gold, silicon dioxide, and mica is higher than the adhesion of silicon dioxide probes on the same substrates.

The hydrolysis of new lipid-like substrates and trilaurin in monolayers catalyzed with the lipase fromPseudomonas fluorescens

A simple method for determining the enzymic hydrolysis parameters of lipid-like substrates and trilaurin assembled in monolayers at the water-air interface was suggested. At a surface pressure of 10 mN/m, the initial rates of lipolysis were found to be proportional to the decrease in area of the substrate monolayer caused by the enzymic hydrolysis in a single-compartment Langmuir balance. The kinetic parameters for the hydrolysis of trilaurin and three 1,3-dilaurylpseudoglycerides acetylated in position 2 with an amino acid (phenylalanine, leucine, or valine) catalyzed with lipase fromPseudomonas fluorescens were determined. Unlike models of enzymic hydrolysis that neglect the thickness of the substrate monolayer, our method allows the determination of kinetic parameters in standard dimensions. The values ofkcat for the synthetic pseudoglycerides were found to be significantly higher than that for trilaurin, while the values ofKm(app) were close. This may be due to the presence of positively charged primary amino groups in the molecules of pseudoglycerides.