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Because nanoparticles are finding uses in myriad biomedical applications, including the delivery of nucleic acids, a detailed knowledge of their interaction with the biological system is of utmost importance. Here the size-dependent uptake of gold nanoparticles (AuNPs) (20, 30, 50 and 80 nm), coated with a layer-by-layer approach with nucleic acid and poly(ethylene imine) (PEI), into a variety of mammalian cell lines is studied. In contrast to other studies, the optimal particle diameter for cellular uptake is determined but also the number of therapeutic cargo molecules per cell. It is found that 20 nm AuNPs, with diameters of about 32 nm after the coating process and about 88 nm including the protein corona after incubation in cell culture medium, yield the highest number of nanoparticles and therapeutic DNA molecules per cell. Interestingly, PEI, which is known for its toxicity, can be applied at significantly higher concentrations than its IC(50) value, most likely because it is tightly bound to the AuNP surface and/or covered by a protein corona. These results are important for the future design of nanomaterials for the delivery of nucleic acids in two ways. They demonstrate that changes in the nanoparticle size can lead to significant differences in the number of therapeutic molecules delivered per cell, and they reveal that the toxicity of polyelectrolytes can be modulated by an appropriate binding to the nanoparticle surface.

Upon slow crystallization from silica-containing solutions or gels at elevated pH, alkaline-earth carbonates spontaneously self-assemble into remarkable nanocrystalline ultrastructures. These so-called silica biomorphs exhibit curved morphologies beyond crystallographic symmetry and ordered textures reminiscent of the hierarchical design found in many biominerals. The formation of these fascinating materials is thought to be driven by a dynamic coupling of the components' speciations in solution, which causes concerted autocatalytic mineralization of silica-stabilized nanocrystals over hours. In the present work, we have studied the precipitation kinetics of this unique system by determining growth rates of individual aggregates using video microscopy, and correlated the results with time-dependent data on the concentration of metal ions and pH acquired online during crystallization. In this manner, insight to the evolution of chemical conditions during growth was gained. It is shown that crystallization proceeds linearly with time and is essentially reaction controlled, which fits well in the proposed morphogenetic scenario, and thus, indirectly supports it. Measurements of the silica concentration in solution, combined with analyses of crystal aggregates isolated at distinct stages of morphogenesis, further demonstrate that the fraction of silica coprecipitated with carbonate during active growth is rather small. We discuss our findings with respect to the role of silica in the formation of biomorphs, and moreover, prove that the external silica skins that occasionally sheath the aggregates--previously supposed to be involved in the growth mechanism--originate from secondary precipitation after growth is already terminated.

Nanoarchaeum equitans und Ignicoccus hospitalis sind anaerobe hyperthermophile Mikroorganismen. Sie reprasentieren eine einzigartige, rein archaeelle Lebensgemeinschaft, in der N. equitans zwingend auf seinen Wirt I. hospitalis angewiesen ist, da es alle seine Lipide und Aminosauren von ihm bezieht. I. hospitalis lebt von der Reduktion elementaren Schwefels mit Wasserstoff. Er besitzt einen neuen CO2-Fixierungsweg und eine fur Archaeen einmalige Zellstruktur aus innerer und auserer Membran, die zwei Zellkompartimente bilden, das Cytoplasma und einen grosen Intermembranraum. Durch immunologische Analysen zeigten wir, dass bei I. hospitalis die ATP-Synthase- und die H2:Schwefel-Oxidoreduktase-Komplexe in seiner auseren Membran lokalisiert sind. Damit ist er der erste Prokaryot mit einer energetisierten auseren Membran, ATP-Synthese auserhalb des Cytoplasmas und der raumlichen Trennung von Energiekonservierung und Informationsubertragung oder Proteinbiosynthese. Daraus ergeben sich zahlreiche Fragen zur Funktion und Charakterisierung der beiden Membranen und Zellkompartimente sowie einem moglichen Energietransfer zu N. equitans. An enigmatical association of two Archaea The obligate anaerobic hyperthermophilic Nanoarchaeum equitans and Ignicoccus hospitalis represent a unique, purely archaeal biocoenosis which is mandatory for N. equitans. Its strong dependence on I. hospitalis is affirmed by the fact that its lipids and amino acids are obtained exclusively from the host. The Crenarchaeon I. hospitalis is characterized by energy production via reduction of elemental sulfur with molecular hydrogen and a novel CO2-fixation pathway. It possesses a unique cell envelope for Archaea with an inner and an outer membrane, forming two cell compartments, the cytoplasm and a huge intermembrane compartment. By immuno-analyses we demonstrated that the ATP synthase and H2:sulfur oxidoreductase complexes of I. hospitalis are located in the outer membrane. Thus I. hospitalis is the first Prokaryote with an energized outer membrane, ATP synthesis outside the cytoplasm, and spatial separation of energy conservation from information processing and protein biosynthesis. This raises many questions on the function and characterization of the two membranes, the two cell compartments, and a possible ATP transfer to N. equitans.

The cell walls of Sulfolobales species consist of proteinaceous S-layers assembled from two polypeptides, SlaA and SlaB. We isolated the large S-layer protein of Acidianus ambivalens and both S-layer subunits of Sulfolobus solfataricus and Metallosphaera sedula, respectively. The slaAB genes, lying adjacently in the chromosomes, are constitutively transcribed as bicistronic operons in A. ambivalens and S. solfataricus. A smaller slaA transcript appeared in Northern hybridizations of A. ambivalens RNA. PCRs experiments showed that 80-85% of the transcripts stop at an oligo-T terminator downstream of slaA while 15-20% are read through to a second terminator downstream of slaB. The bicistronic operons including promoter and terminator regions are conserved in the Sulfolobales. While no SlaA homologue is found outside the Sulfolobales, SlaB is distantly similar to S-layer proteins of other Crenarchaeota, e.g. the Staphylothermus marinus tetrabrachion. Molecular modelling suggests SlaBs to be composed of 2-3 consecutive beta sandwich domains, a coiled-coil domain of 15-17 nm in length and a C-terminal transmembrane helix. Electron microscopy shows crystalline protein arrays with triangular and hexagonal pores. We propose that the mushroom-shaped 'unit cells' of the Sulfolobales' S-layers consist of three SlaBs anchoring the complex in the membrane and six SlaAs forming the detergent-resistant outer sacculus.

Enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11) from the hyperthermophilic bacterium Thermotoga maritima was purified to homogeneity. The N-terminal 25 amino acids of the enzyme reveal a high degree of similarity to enolases from other sources. As shown by sedimentation analysis and gel-permeation chromatography, the enzyme is a 345-kDa homoctamer with a subunit molecular mass of 48 +/- 5 kDa. Electron microscopy and image processing yield ring-shaped particles with a diameter of 17 nm and fourfold symmetry. Averaging of the aligned particles proves the enzyme to be a tetramer of dimers. The enzyme requires divalent cations in the activity assay, Mg2+ being most effective. The optimum temperature for catalysis is 90 degrees C, the temperature dependence yields a nonlinear Arrhenius profile with limiting activation energies of 75 kJ mol-1 and 43 kJ mol-1 at temperatures below and above 45 degrees C. The pH optimum of the enzyme lies between 7 and 8. The apparent Km values for 2-phospho-D-glycerate and Mg2+ at 75 degrees C are 0.07 mM and 0.03 mM; with increasing temperature, they are decreased by factors 2 and 30, respectively. Fluoride and phosphate cause competitive inhibition with a Ki of 0.14 mM. The enzyme shows high intrinsic thermal stability, with a thermal transition at 90 and 94 degrees C in the absence and in the presence of Mg2+.

The ability to adhere onto surfaces is of very high importance for microorganisms, enabling them to stay in a favourable habitat for life. In the case of Bacteria cell surface organelles called fimbriae/pili have been shown to be used for adhesion; corresponding cell surface appendages of Archaea have not yet been defined. The first detailed characterization of archaeal fimbriae, namely those of Methanothermobacter thermoautotrophicus, allowed us to identify mth60 as the main structural fimbrin gene. Recombinant expression of mth60 in Escherichia coli was used to generate sufficient amounts of Mth60 to induce antibodies in rabbits. The antiserum reacted specifically with the 16 kDa fimbrial glycoprotein and could specifically detach adhering M. thermoautotrophicus cells from various surfaces. In addition we proved that cells adhering to solid surfaces - organic and inorganic ones - express many more fimbriae than cells growing in liquid cultures. The Mth60 fimbriae therefore are used by M. thermoautotrophicus as adhesins.

Proteinaceous, hair-like appendages known as fimbriae or pili commonly extend from the surface of prokaryotic cells and serve important functions such as cell adhesion, biofilm formation, motility and DNA transfer. Here we show that a novel group of archaea from cold, sulphidic springs has developed cell surface appendages of an unexpectedly high complexity with a well-defined base-to-top organization. It represents a new class of filamentous cell appendages, for which the term 'hamus' is proposed. Each archaeal cell is surrounded by a halo of about 100 hami, which mediate strong adhesion of the cells to surfaces of different chemical composition. The hami are mainly composed of 120 kDa subunits and remained stable in a broad temperature and pH range (0-70 degrees C; 0.5-11.5). Electron microscopy and cryo-electron tomography revealed that the hamus filament possesses a helical basic structure. At periodic distances, three prickles emanate from the filament, giving it the character of industrially produced barbwire. At its distal end the hami carry a tripartite, barbed grappling hook (60 nm in diameter). The architecture of this molecular hook is reminiscent of man-made fishhooks, grapples and anchors. It appears that nature has developed a perfect mechanical nano-tool in the course of biological evolution, which also might prove useful in the field of nanobiotechnology.

The covalently linked cell wall protein Ccw12p of Saccharomyces cerevisiae is a GPI-anchored protein (V. Mrsa et al., 1999, J Bacteriol 181: 3076-3086). Although only 121 amino acids long, the haemagglutinin-tagged protein released by laminarinase from the cell wall possesses an apparent molecular mass of > 300 kDa. A membrane-bound form with an apparent molecular mass of 58 kDa is highly O- and N-glycosylated and contains the GPI anchor. With a half-life of 2 min, the membrane form is transformed to the > 300 kDa form. The deletion mutant ccw12Delta grows slower than the wild type, is highly sensitive to Calcofluor white and contains 2.5 times more chitin. Further, compared with wild-type yeast, significantly more proteins are released from intact cells when treated with dithiothreitol. Interestingly, these defects become less pronounced when further GPI-anchored cell wall proteins are deleted. Mutant DeltaGPI (simultaneous deletion of CCW12, CCW13/DAN1, CCW14, TIP1 and CWP1) is similar in many respects to wild-type yeast. To find out how the cell wall is stabilized in mutant DeltaGPI, a genome-wide transcription analysis was performed. Of 159 significantly regulated genes, 14 encode either known or suspected cell wall-associated proteins. Analysis of genes affected in transcription revealed that SED1 and SRL1 in particular are required to reconstruct cell wall stability in the absence of multiple GPI-anchored mannoproteins.

Mating type-specific agglutination of Saccharomyces cerevisiae a and alpha cells depends on the heterophilic interaction of two cell surface glycoproteins, the gene products of AG alpha 1 and AGA2. Evidence is presented with immunogold labelling that the alpha-agglutinin is part of the outer fimbrial cell wall coat. The a-agglutinin is bound via two S-S bridges (Cys7 and Cys50) to a cell wall component, most probably the gene product of AGA1. His273 of alpha-agglutinin has previously been shown to be essential for a- and alpha-agglutinin interaction and a model based on two opposing ion-pairs had been proposed. By site-directed mutagenesis this possibility has now been excluded. With the help of various peptides, either chemically synthesized, obtained by proteolysis of intact glycosylated a-agglutinin or prepared from a fusion protein expressed in Escherichia coli, the biologically active region of a-agglutinin was located at the C-terminus of the molecule. A peptide consisting of the C-terminal 10 amino acids (GSPIN-TQYVF) was active in nanomolar concentrations. Saccharide moieties, therefore, are not essential for the mating type-specific cell-cell interaction; glycosylated peptides are, however, four to five times more active than non-glycosylated ones. Comparisons of the recognition sequences of the S. cerevisiae agglutinins with that of the Dictyostelium contact site A glycoprotein (gp80), as well as with those of the various families of cell adhesion molecules of higher eucaryotes, have been made and are discussed.

SUMMARY A rapid cooling/cryotransfer system was designed to achieve a high reproducibility in vitrifying thin water films containing biological specimens. In order to improve the contrast these unstained specimens were deliberately freeze-dried in situ in the electron microscope. The preservation of the structure obtained by freeze-drying at 180 K and subsequent microscopy at 90 K is very encouraging as has been shown with two types of test specimen. Tubular photosynthetic membranes were used to examine the effects of a variety of freeze-drying conditions on structural preservation, as judged by flattening of the tubes. A two-dimensional protein crystal was used to evaluate problems of low-dose microscopy of unstained, freeze-dried proteins, e.g. optical density of films, motif detection by cross-correlation and transferring the accurate molecular positions from medium-dose to corresponding low-dose micrographs. The radiation sensitivity of the unstained, freeze-dried protein crystal was investigated by a dose series covering a wide range.