Bacterial IscU is a well folded and functional single domain protein.

Iron-sulfur clusters are widely represented in most organisms, but the mechanism of their formation is not fully understood. Of the two main proteins involved in cluster formation, NifS/IscS and NifU/IscU, only the former has been well studied from a structural point of view. Here we report an extensive structural characterization of Escherichia coli IscU. We show by a variety of physico-chemical techniques that E. coli IscU construct can be expressed to high purity as a monomeric protein, characterized by an alphabeta fold with high alpha-helix content. The high melting temperature and the reversibility of the thermal unfolding curve (as measured by CD spectroscopy) hint at a well ordered stable fold. The excellent dispersion of cross peaks in the (1)H-(15)N correlation spectrum is consistent with these observations. Monomeric E. coli IscU is able to provide a scaffold for Iron-sulfur cluster assembly, but has no direct interaction with either Fe(II) or Fe(III) ions, suggesting the need of further partners to achieve a stable interaction.     

The missing links to link ubiquitin: Methods for the enzymatic production of polyubiquitin chains

Attachment of ubiquitin (Ub) as monoUb and polyUb chains of different lengths and linkages to proteins plays a dominant role in very different regulatory mechanisms. Therefore, the study of polyUb chains has assumed a central interest in biochemistry and structural biology. An essential step necessary to allow in vitro biochemical and structural studies of polyUbs is the production of their chains in high quantities and purity. This is not always an easy task and can be achieved both enzymatically and chemically. Previous reviews have covered chemical cross-linking exhaustively. In this review, we concentrate on the different approaches developed so far for the enzymatic production of different Ub chains. These strategies permit a certain flexibility in the production of chains with various linkages and lengths. We critically describe the available methods and comment on advantages and limitations. It is clear that the field is mature to study most of the possible links, but some more work needs to be done to complete the picture and to exploit the current methodologies for understanding in full the Ub code.     

The methodological approach for the generation of humandendritic cells from monocytes affects the maturation state of the resultant dendritic cells

Dendritic cells (DCs) are effective as antigen-presenting cells in the immune system and are present at two functional stages depending on their maturation state. For experimental investigation of this concept, CD14⁺ monocytes from blood are isolated and cultured to generate in vitro the DCs needed for functional analysis. For positive selection of CD14⁺ monocytes we compared two immunomagnetic bead technologies: MACS^® Separation, created by Miltenyi Biotec, and EasySep^® Selection, created by StemCell Technologies. The monocytes provided dendritic cells for their functional analysis. Lipopolysaccharide was added to cultured DCs to induce maturation. Although both systems generated DCs from the positively selected CD14⁺ cells, there were certain differences between them. Morphological, phenotypic, and functional analysis showed that MACS^®-selection provided DCs that have typical features corresponding to day 6 or 7 of maturation. EasySep^®–DCs exist in a partially-mature state from day 6 onward, even without the addition of a maturation stimulus. The reason behind this partial maturation is possibly based on the dextran-coated beads that are associated with the EasySep^® product. Both methods provide pure and viable DCs, but we would recommend using the MACS^® system for obtaining DCs suitable for functional studies.     

Effects of iron on the aggregation propensity of the N-terminal fibrillogenic polypeptide of human apolipoprotein A-I

Specific mutations in APOA1 gene lead to systemic, hereditary amyloidoses. In ApoA-I related amyloidosis involving the heart, amyloid deposits are mainly constituted by the 93-residue N-terminal region of the protein, here indicated as [1-93]ApoA-I. Oxidative stress is known to be an enhancing factor for protein aggregation. In healthy conditions, humans are able to counteract the formation and the effects of oxidative molecules. However, aging and atmospheric pollution increase the concentration of oxidative agents, such as metal ions. As the main effect of iron deregulation is proposed to be an increase in oxidative stress, we analysed the effects of iron on [1-93]ApoA-I aggregation. By using different biochemical approaches, we demonstrated that Fe(II) is able to reduce the formation of [1-93]ApoA-I fibrillar species, probably by stabilizing its monomeric form, whereas Fe(III) shows a positive effect on polypeptide fibrillogenesis. We hypothesize that, in healthy conditions, Fe(III) is reduced by the organism to Fe(II), thus inhibiting amyloid formation, whereas during ageing such protective mechanisms decline, thus exposing the organism to higher oxidative stress levels, which are also related to an increase in Fe(III). This alteration could contribute to the pathogenesis of amyloidosis.     

Distribution of DNA methylation,CpGs, and CpG islands in human isochores

DNA methylation is a major epigenetic modification of the genome that affects basic biological functions, such as gene expression and cell development. We used the human genome sequences and the DNA methylation data that are available in order to establish a map of the levels of GC and methylation in isochores. We also looked for the correlations that hold between GC levels and the distribution of the (1) dinucleotide CpG, (2) ratio 5mC/CpG, and (3) CpG islands. Our results show that methylation levels, CpG frequencies, and the density of CpG islands are positively correlated with the GC level of isochores. In contrast, the correlation between the 5mC/CpG ratio and GC is a negative one because the increase in methylation lags behind that of CpG, to reach a plateau in the GC-richest, gene-richest isochore families H2 and H3. In conclusion, there are more CpG targets that remain unmethylated in the GC-richest, gene-richest isochores in comparison with the other isochores. This conclusion supports the idea that the widespread methylation under consideration here has a general inhibitory effect on gene expression.     

Purification and Characterization of Type 1 Fimbriae of Salmonella typhi

Type 1 fimbriae have been purified from a Salmonella typhi strain of clinical origin. Purified fimbriae retained their ability to bind to erythrocytes in a mannose-inhibitable fashion and, in doing so, behaved preferentially as a monovalent adhesin. SDS-PAGE analysis of the fimbrial preparation showed the presence of a 20-kDa major polypeptide component (fimbrillin) and of additional larger polypeptides present in smaller amounts. The amino-terminal sequence of fimbrillin was determined and turned out to be very similar but not identical to that of type 1 fimbrillins of other Salmonella serovars. A Western blot analysis of the purified fimbrial preparation using an antiserum raised against native fimbriae suggested that fimbrial proteins did not carry any major sequential epitope and that, in native fimbriae, conformational epitopes, possibly generated between different subunits, might provide for the major immunogenic epitopes. Analysis of different S. typhi clinical isolates using the anti-fimbrial antiserum showed an overall immunological similarity of these structures within this serovar.     

Comparative anatomy and evolutionary history of suction feeding in cetaceans

Some odontocetes possess unique features of the hyolingual apparatus that are involved in suction feeding. The hyoid bone and associated musculature generates rapid, piston‐like retraction, and depression of the hyoid and tongue. “Capture” suction feeders (e.g., Globicephala) use suction for capturing and swallowing prey. “Combination” feeders (i.e., Lagenorhynchus) use both raptorial feeding (to capture prey) and suction (to ingest prey). In “capture” suction feeders, features of the hyoid and skull have been attributed to creating suction (i.e., large surface area and mandibular bluntness). In addition to odontocetes, a mysticete, the gray whale (Eschrichtius robustus), is considered a benthic suction feeder. However, anatomical studies of purported suction‐feeding structures of the gray whale are lacking. In addition, few studies have utilized evolutionary approaches to understand the history of suction feeding in cetaceans. This study incorporates quantitative and qualitative hyoid and cranial data from 35 extant and 14 extinct cetacean species into a multivariate principal component analysis and comparative phylogenetic analyses. Conclusions from these analyses are that some commonly attributed features (i.e., ventral throat grooves and mandibular bluntness) and one principal component are significantly correlated with suction feeding. Finally, ancestral state reconstructions indicate that suction feeding likely evolved once, early in cetacean evolutionary history.     

Interaction between nanomaterials and the innate immune system across evolution

Interaction of engineered nanomaterials (ENMs) with the immune system mainly occurs with cells and molecules of innate immunity, which are present in interface tissues of living organisms. Immuno-nanotoxicological studies aim at understanding if and when such interaction is inconsequential or may cause irreparable damage. Since innate immunity is the first line of immune reactivity towards exogenous agents and is highly conserved throughout evolution, this review focuses on the major effector cells of innate immunity, the phagocytes, and their major sensing receptors, Toll-like receptors (TLRs), for assessing the modes of successful versus pathological interaction between ENMs and host defences. By comparing the phagocyte- and TLR-dependent responses to ENMs in plants, molluscs, annelids, crustaceans, echinoderms and mammals, we aim to highlight common recognition and elimination mechanisms and the general sufficiency of innate immunity for maintaining tissue integrity and homeostasis.