Direct measurement of the mechanism by which magnesium specifically modifies the mechanical properties of DNA.

We examine the effect of physiological cations Na+, K+, Mg2+, and Ca2+ on the mechanical properties of bundles of λ-phage DNA using silicon nanotweezers (SNTs). Integrating SNTs with a microfluidic device allows us to perform titration experiments while measuring the effect in real-time. The results show that only for Mg2+ and in particular, at the intra-nuclear concentration (100 mM), the interaction occurs.

Structural requirements for anti-oxidant activity of calix[n]arenes and their associated anti-bacterial activity.

Treatment of neural cells with calix[n]arenes featuring sulphonate moieties and linked to Ag nanoparticles results in reduced reactive species. For Gram+ bacteria there is an inverse correlation between anti-bacterial activity and ROS reduction whereas for Gram- bacteria only calix[6]arenes bearing O-alkyl sulphonate functions act as ROS inhibitors and anti-bacterial agents.

A COMPARATIVE ANALYSIS OF THE VOLVOCACEAE (CHLOROPHYTA)(1).

This review covers essentially all aspects of the organisms in the green algal family Volvocaceae and suggests the genetic history of the various steps in their evolution from their unicellular ancestors.

Core-shell Au@(TiO(2), SiO(2)) nanoparticles with tunable morphology.

A novel approach based on the Stöber method allows breaking of the symmetry of core-shell systems based on metallic core and metal oxide shell. By adjusting the proportion of the TiO(2) precursor with regard to the silica precursor, different morphologies of the particles have been obtained displacing the gold particle from center to eccentric positions leading to acorn-like and raspberry-like structure.

Paramecium aurelia revisited.

The species Paramecium aurelia sensu latu, containing 15 sexually isolated subspecies (syngens), is the classic example of a sibling species complex in the ciliates. Using DNA sequence comparison, it is now possible to see whether this example parallels other studied sibling species complexes. We sequenced the internal transcribed spacer (ITS) region of the nuclear ribosomal cistron for 13 of the syngens plus two other Paramecium species and several Tetrahymena spp. Using available spirotrich sequences of the internal transcribed spacer 2 (ITS2), we established the RNA transcript folding pattern for ciliates. Ciliates exhibit the two highly conserved helices in their RNA transcript folding pattern in common with other eukaryotes, despite their unusual nuclear behavior and their presumed low copy number of micronuclear ribosomal repeats. Consequently, the set of 111-116 ITS2 nucleotide positions that are relatively conserved in evolution can be derived and used for comparative analysis. Mating behavior (i.e. gamete agglutination and fusion) is the character showing greatest correlation with the degree of ITS2 evolution in the P. aurelia complex, as also found in other eukaryotes. The degree of change in the ITS2 relatively conserved sequences found among the sibling species of P. aurelia is the same degree as found among the sibling species of the Drosophila melanogaster-mauritania-sechellia-simulans-yakuba species complex. The relatively conserved subregion of ITS2, determined from transcript secondary structure, is a tool for identifying the level of the biological species in the absence of knowledge of sexual compatibility in both micro- and macro-eukaryote species complexes.

Atomic force microscopy imaging of novel type of polymeric colloidal nanostructures.

Polymeric nanoparticles were prepared by the interfacial poly-condensation of the lipophilic monomer, phtaloyldichloride and the hydrophilic monomer, diethylenetriamine, in the presence and absence of the surfactant Pluronic F68. The colloidal systems were analysed by dynamic light scattering and atomic force microscopy, the structures formed have two populations (150 and 350 nm) in the presence of the surfactant and one population (450 nm) in the absence of the surfactant. The results can be interpreted in terms of the formation of hollow nanocapsules that collapse on deposition and drying.

NADPH oxidase of Epstein-Barr-virus immortalized B lymphocytes. Effect of cytochrome b(558) glycosylation.

The phagocyte NADPH oxidase is known to be expressed in Epstein-Barr virus (EBV) immortalized B lymphocytes. But even if its molecular composition and its catalytic mechanisms are similar, the activity measured in B cells is very low compared to that of neutrophils. This could be explained by the low expression of cytochrome b558, the membrane redox component, but also by a defect in the activation process. This work is focused on gp91-phox glycosylation in B lymphocytes to assess its role in the complex assembly upon activation. Atomic force microscopy (AFM) combined with immunochemical approaches were used to investigate the effect of the glycosylation on the structure of cytochrome b558 inserted into liposomes, on the reconstituted oxidase activity in vitro, and to directly monitor interaction forces between specific antibodies and the hemoprotein in its native or deglycosylated state. The results show that in EBV-B cells, gp91-phox glycosylation is higher than in neutrophils. The interaction force measured between the monoclonal antibody 11C12, known to inhibit O(-2) production in B lymphocytes, and the hemoprotein is increased after deglycosylation. This suggested that the epitope region recognized by this antibody is partly hidden in B cells, and that this region could be involved in the conformational change that occurs in the hemoprotein during the complex assembly. The high glycosylation of gp91-phox in B cells associated with the lipidic environment could lead to additional structural constraints in the membrane-bound hemoprotein that partly blocked the hemoprotein in its inactive state.

Protein-calixarene interactions: complexation of Bovine Serum Albumin by sulfonatocalix[n]arenes.

The complexation of Bovine Serum Albumin with sulfonatocalix[n]arenes has been demonstrated by means of electrospray mass spectrometry, dynamic light scattering and atomic force microscopy; with sulfonatocalix[4]arene one strong and two weaker binding sites are detected; the effects on the structure of thin films formed by surface deposition of BSA show that the sulfonatocalix[n]arenes act to reticulate the films and produce essentially planar systems.

Hydroxylated nanoballs: synthesis, crystal structure, solubility and crystallization on surfaces.

The reaction of equimolar amounts of Cu(NO3)2 and bdc-5-OH (bdc-5-OH = benzene-1,3-dicarboxylate-5-hydroxy) affords hydroxylated nanoballs with high solubility and an ability to form microcrystals on surfaces.

p-Sulfonatocalix[6]arene is an effective coacervator of poly(allylamine hydrochloride).

p-Sulfonatocalix[6]arene is shown to form insoluble complexes with poly(allylamine hydrochloride) when the charge balance between the negative calixarene sulfonate groups matches the positive charge carried by the polyelectrolyte, this makes this glycosylaminoglycan analog an interesting candidate for controlled release systems in the case of proteins encapsulated in mesoscopic complexes with polyelectrolytes.

P67-phox-mediated NADPH oxidase assembly: imaging of cytochrome b558 liposomes by atomic force microscopy.

NADPH oxidase activity depends on the assembly of the cytosolic activating factors, p67-phox, p47-phox, p40-phox, and Rac with cytochrome b(558). The transition from an inactive to an active oxidase complex induces the transfer of electrons from NADPH to oxygen through cytochrome b(558). The assembly of oxidase complex was studied in vitro after reconstitution in a heterologous cell-free assay by using true noncontact mode atomic force microscopy. Cytochrome b(558) was purified from neutrophils and Epstein-Barr virus-immortalized B lymphocytes and incorporated into liposomes. The effect of protein glycosylation on liposome size and oxidase activity was investigated. The liposomes containing the native hemoprotein purified from neutrophils had a diameter of 146 nm, whereas after deglycosylation, the diameter was reduced to 68 nm, although oxidase activity was similar in both cases. Native cytochrome b(558) was used after purification in reconstitution experiments to investigate the topography of NADPH oxidase once it was assembled. For the first time, atomic force microscopy illustrated conformational changes of cytochrome b(558) during the transition from the inactive to the active state of oxidase; height measurements allow the determination of a size of 4 nm for the assembled complex. In the processes that were studied, p67-phox displayed a critical function; it was shown to be involved in both assembly and activation of oxidase complex while p47-phox proceeded as a positive effector and increased the affinity of p67-phox with cytochrome b(558), and p40-phox stabilizes the resting state. The results suggest that although an oligomeric structure of oxidase machinery has not been demonstrated, allosteric regulation mechanisms may be proposed.

Phylogenetic analysis of "Volvocacae" for comparative genetic studies.

Sequence analysis based on multiple isolates representing essentially all genera and species of the classic family Volvocaeae has clarified their phylogenetic relationships. Cloned internal transcribed spacer sequences (ITS-1 and ITS-2, flanking the 5.8S gene of the nuclear ribosomal gene cistrons) were aligned, guided by ITS transcript secondary structural features, and subjected to parsimony and neighbor joining distance analysis. Results confirm the notion of a single common ancestor, and Chlamydomonas reinharditii alone among all sequenced green unicells is most similar. Interbreeding isolates were nearest neighbors on the evolutionary tree in all cases. Some taxa, at whatever level, prove to be clades by sequence comparisons, but others provide striking exceptions. The morphological species Pandorina morum, known to be widespread and diverse in mating pairs, was found to encompass all of the isolates of the four species of Volvulina. Platydorina appears to have originated early and not to fall within the genus Eudorina, with which it can sometimes be confused by morphology. The four species of Pleodorina appear variously associated with Eudorina examples. Although the species of Volvox are each clades, the genus Volvox is not. The conclusions confirm and extend prior, more limited, studies on nuclear SSU and LSU rDNA genes and plastid-encoded rbcL and atpB. The phylogenetic tree suggests which classical taxonomic characters are most misleading and provides a framework for molecular studies of the cell cycle-related and other alterations that have engendered diversity in both vegetative and sexual colony patterns in this classical family.

Intraspecies analysis: comparison of ITS sequence data and gene intron sequence data with breeding data for a worldwide collection of Gonium pectorale.

The morphologically uniform species Gonium pectorale is a colonial green flagellate of worldwide distribution. The affinities of 25 isolates from 18 sites on five continents were assessed by both DNA sequence comparisons and sexual compatibility. Complete sequences were obtained (i) for the internal transcribed spacer ITS-1 and ITS-2 regions of ribosomal DNA and (ii) for each of three single-copy spliceosomal introns, two in a small G protein and one in the actin gene. ITS sequences appeared to homogenize sufficiently rapidly to behave as a single copy gene. Intron sequence differences between isolates in this species reached nucleotide substitution saturation, while ITS sequences did not. Parsimony and evolutionary distance analysis of the two types of DNA data gave essentially the same tree conformation. By all these criteria, the group of G. pectorale isolates fell into two main clades, A and B. Clade A, with isolates from four continents, was comprised of four subclades of quite closely related isolates, plus one strain of ambiguous affinity. Clade B was comprised of two subclades represented by South African and South American isolates, respectively; thus, only subclades of clade B showed geographical localization. With respect to mating, all isolates except one homothallic strain and one apparently sterile strain fell into either one or the other of two mating types. Pairings in all possible combinations revealed that isolates from the same site formed abundant zygotes, which germinated to produce new, sexually active organisms. Zygotes were also formed in many pairings of other combinations, including crosses of clade A with clade B organisms, but none of the latter produced viable germlings. The ability to mate and produce viable progeny that were themselves capable of sexual reproduction was restricted to members of subclades established on the basis of DNA sequence similarities. Thus, the grades of difference in both nuclear intron sequences and rDNA ITS sequences paralleled those observed in the sexual analysis.

Derivation of the Secondary Structure of the ITS-1 Transcript in Volvocales and its Taxonomic Correlations.

Knowledge of secondary structure, formed by the gene spacer regions of the primary transcript of nuclear rDNA cistrons, is lacking for most phyla of eukaryotes. We have sequenced the first internal transcribed spacer region (ITS-1) of multiple representatives of the Volvocales, and from comparisons of these, derived a secondary structure common to the entire group. The secondary structure model is supported by numerous compensating base pair changes located within the paired regions of the stem-loops. Within the morphological species, such as those of Astrephomene and Gonium, the three basal nucleotide pairs of helices are highly conserved in primary sequence, and the single stranded region rich in CCAA is identical in sequence, even when isolates come from all continents of the earth. In other Volvocacean species known to include many pairs of mating types, this same level of conservation is found to correlate with the mating subgroups of the species. Thus a comparable degree of sequence similarity appears to characterize all isolates of a "biological" species; this is valid for taxonomic species only where the biological and taxonomic species levels coincide. In addition, the ITS-1 contains information useful for population analyses, and spacer secondary structure may have additional phylogenetic utility at the level of class or subclass when that information becomes available for other protistan groups.

Ribosomal DNA ITS-1 and ITS-2 sequence comparisons as a tool for predicting genetic relatedness.

The determination of the secondary structure of the internal transcribed spacer (ITS) regions separating nuclear ribosomal RNA genes of Chlorophytes has improved the fidelity of alignment of nuclear ribosomal ITS sequences from related organisms. Application of this information to sequences from green algae and plants suggested that a subset of the ITS-2 positions is relatively conserved. Organisms that can mate are identical at all of these 116 positions, or differ by at most, one nucleotide change. Here we sequenced and compared the ITS-1 and ITS-2 of 40 green flagellates in search of the nearest relative to Chlamydomonas reinhardtii. The analysis clearly revealed one unique candidate, C. incerta. Several ancillary benefits of the analysis included the identification of mislabelled cultures, the resolution of confusion concerning C. smithii, the discovery of misidentified sequences in GenBank derived from a green algal contaminant, and an overview of evolutionary relationships among the Volvocales, which is congruent with that derived from rDNA gene sequence comparisons but improves upon its resolution. The study further delineates the taxonomic level at which ITS sequences, in comparison to ribosomal gene sequences, are most useful in systematic and other studies.

The internal transcribed spacer 2 exhibits a common secondary structure in green algae and flowering plants.

Sequences of the Internal Transcribed Spacer 2 (ITS-2) regions of the nuclear rDNA repeats from 111 organisms of the family Volvocaceae (Chlorophyta) and unicellular organisms of the Volvocales, including Chlamydomonas reinhardtii, were determined. The use of thermodynamic energy optimization to generate secondary structures and phylogenetic comparative analysis of the spacer regions revealed a common secondary structure that is conserved despite wide intra- and interfamilial primary sequence divergence. The existence of this conserved higher-order structure is supported by the presence of numerous compensating basepair changes as well as by an evolutionary history of insertions and deletions that nevertheless maintains major aspects of the overall structure. Furthermore, this general structure is preserved across broad phylogenetic lines, as it is observed in the ITS-2s of other chlorophytes, including flowering plants; previous reports of common ITS-2 secondary structures in other eukaryotes were restricted to the order level. The reported ITS-2 structure possesses important conserved structural motifs which may help to mediate cleavages in the ITS-2 that occur during rRNA transcript processing. Their recognition can guide further studies of eukaryotic rRNA processing, and their application to sequence alignments may contribute significantly to the value of ITS-2 sequences in phylogenetic analyses at several taxonomic levels, but particularly in characterizing populations and species.

Synthesis and immunostimulating properties of lipophilic ester and ether muramyl peptide derivatives.

Macrophages can become cytotoxic toward tumor cells when activated by immunomodulators. Three different muramyl peptides were synthesized: one hydrolyzable lipophilic ester derivative (MTP-Chol) and two nonhydrolyzable lipophilic ether derivatives (MTP-octadecane and MTP-heptadecafluorooctadecane). Activation of the RAW 264.7 cell line was studied by measuring nitrite production as an indication of NO-synthase activity. The lipophilic ester derivative, incorporated within nanocapsules, was as active as free muramyl dipeptide, whereas the lipophilic ether derivatives were unable to activate macrophages. MTP-octadecane in micellar form was not capable of inducing macrophage cytotoxicity either. These results indicate that lipophilic muramyl peptides need to be hydrolyzed to yield a hydrosoluble metabolite in order to activate macrophages.

The crystal structure of 6I-(6-aminohexyl)amino-6I-deoxycyclomaltoheptaose.

The monosubstituted cyclomaltoheptaose derivative, 6I-(6-aminohexyl)amino-6I-deoxycyclomaltoheptaose, crystallizes in the orthorhombic space group P2(1)2(1)2(1) with a = 32.513(2), b = 15.3871(9), c = 15.2645(9) A, V = 7636.6(8) A3 and Z = 4. The macrocycles are spirally aligned along the twofold screw axis parallel to the c crystal axis forming polymeric-like columns. The 6-aminohexyl chain enters the cavity of an adjacent cyclomaltoheptaose moiety in the column from the secondary side and its extremity protrudes from the primary side of the latter. All the atoms of the chain exhibit high thermal motion.

Fate of Parasite and Host Organelle DNA during Cellular Transformation of Red Algae by Their Parasites.

The transfer of a nucleus into a cytoplasm of a genetically foreign cell and its subsequent multiplication in the cytoplasm of this cell characterize most parasitic red algal species and their interactions with specific red algal hosts. Nuclei enter the host's cytoplasm upon cell fusion of parasite and host cell; here, they replicate, are spread to contiguous host cells, and ultimately are packaged into spores that reinfect other host thalli. In this study, we examined whether the proplastids and mitochondria that occur in these red algal adelphoparasites are acquired from their host or whether they are unique to the parasite and are brought into the host along with the parasite nucleus. To establish their origins and fates, plastid and mitochondrial restriction fragment length polymorphisms (RFLPs) of parasite cells were compared with those of their host plastid and mitochondrial DNA in three host and parasite pairs. For plastids, no RFLP differences were found between hosts and parasites, supporting an earlier conclusion, based on microscopic studies, that the proplastids of parasites are acquired from their hosts. For mitochondria, characteristic RFLP differences were detected between host and parasite for two of the pairs of species but not for the third. Evidence of the evolutionary difference between hosts and their parasites was shown by RFLP differences between nuclear ribosomal repeat regions.