Luca: the last universal common ancestor

One of the most important outcomes of modern biology has been the demonstration of the unity of life. All living beings are in fact descendants of a unique ancestor commonly referred to as Luca (the Last universal common ancestor). The discovery - nearly 30 years ago by Carl Woese - that present-day life on our planet can be assigned to only three domains: two of prokaryotic nature (Archaea and Bacteria), and one eukaryoyic (Eucarya), has given birth to a new field of investigation aimed at determining the nature of Luca. Today, thanks to the accumulation of genomic data, we can loop back into the past and infer a few characters of Luca by comparing what present-day organisms have in common. For example, it is now clear that Luca was a cellular organism provided with a cytoplasmic membrane, and that it harboured already a quite sophisticated translation apparatus. However, the inference of other characters of Luca from comparative genomics is less straightforward: for instance, a few key molecular mechanisms for DNA replication are non-homologous across the three domains and their distribution is often puzzling. This evidence has been embraced by proponents of the hypothesis that Luca harboured an RNA genome and that its replacement by DNA and the appearance of the corresponding molecular systems would have occurred independently in the three life domains after their divergence. However, an equally likely scenario would be that of a Luca with a DNA genome and of a subsequent replacement of its DNA-replication systems by non-homologous counterparts either in the bacterial or in the archaeal/eukaroytic branch. Nevertheless, including the viral world into the picture of the tree of life may thus provide us with precious insights into our most distant past since the invention and spread potential of viruses may have played a key role in early evolution.     

Biocatalysed synthesis of beta-O-glucosides from 9-fluorenon-2-carbohydroxyesters. Part 3: IFN-inducing and anti-HSV-2 properties

In pursuing research on the antiviral, interferon (IFN)-inducing tilorone congeners, a new series of fluoren-carboxyhydroxyesters has been prepared and biologically explored. These esters have subsequently been used as sugar acceptors in the enzymatic transglycosylation reaction using the 'retaining' beta-glycosidase from the archaeon Sulfolobus solfataricus (Ssbeta-Gly). Both aglycones (1-6) and corresponding beta-glucosides (beta-glu 1-beta-glu 6) have been screened for cytotoxicity, interferon-stimulating and antiviral properties against HSV-2. It was found that the addition of compounds beta-glu 5, beta-glu 6 and beta-glu 4 to HSV-2 infected U937 cells downregulates viral replication and triggers cells to release IFN-alpha/beta. Taken together, the results showed improved pharmacological profiles as a consequence of glycosylation. A molecular modelling study carried out on this series of compounds completed the structural characterisation of the novel compounds.     

Nanoarchaea: representatives of a novel archaeal phylum or a fast-evolving euryarchaeal lineage related to Thermococcales?

Background  

Cultivable archaeal species are assigned to two phyla - the Crenarchaeota and the Euryarchaeota - by a number of important genetic differences, and this ancient split is strongly supported by phylogenetic analysis. The recently described hyperthermophile Nanoarchaeum equitans, harboring the smallest cellular genome ever sequenced (480 kb), has been suggested as the representative of a new phylum - the Nanoarchaeota - that would have diverged before the Crenarchaeota/Euryarchaeota split. Confirming the phylogenetic position of N. equitans is thus crucial for deciphering the history of the archaeal domain.     

Visualization through magnetic resonance imaging of DNA internalized following "in vivo" electroporation

The ability to visualize plasmid DNA entrapment in muscle cells undergoing an "in vivo" electroporation treatment was investigated on BALB/c mice using a 7-T magnetic resonance imaging (MRI) scanner using the paramagnetic Gd-DOTA-spd complex as imaging reporter. Gd-DOTA-spd bears a tripositively charged spermidine residue that yields a strong binding affinity toward the negatively charged DNA chain (6.4 kb, K(a) = 2.2 x 10(3) M(-1) for approximately 2500 +/- 500 binding sites). Cellular colocalization of Gd-DOTA-spd and plasmid DNA has been validated by histological analysis of excised treated muscle. In vivo MRI visualization of Gd-DOTA-spd distribution provides an excellent route to access the cellular entrapment of plasmid DNA upon applying an electroporation pulse.     

Temperature-, SDS-, and pH-induced conformational changes in protein disulfide oxidoreductase from the archaeon Pyrococcus furiosus: a dynamic simulation and fourier transform infrared spectroscopic study

The effect of SDS, pD, and temperature on the structure and stability of the protein disulfide oxidoreductase from Pyrococcus furiosus (PfPDO) was investigated by molecular dynamic (MD) simulations and FT-IR spectroscopy. pD affects the thermostability of alpha-helices and beta-sheets differently, and 0.5% or higher SDS concentration influences the structure significantly. The experiments allowed us to detect a secondary structural reorganization at a definite temperature and pD which may correlate with a high ATPase activity of the protein. The MD simulations supported the infrared data and revealed the different behavior of the N and C terminal segments, as well as of the two active sites.     

The unusual intersubunit ferroxidase center of Listeria innocua Dps is required for hydrogen peroxide detoxification but not for iron uptake. A study with site-specific mutants

The role of the ferroxidase center in iron uptake and hydrogen peroxide detoxification was investigated in Listeria innocua Dps by substituting the iron ligands His31, His43, and Asp58 with glycine or alanine residues either individually or in combination. The X-ray crystal structures of the variants reveal only small alterations in the ferroxidase center region compared to the native protein. Quenching of the protein fluorescence was exploited to assess stoichiometry and affinity of metal binding. Substitution of either His31 or His43 decreases Fe(II) affinity significantly with respect to wt L. innocua Dps (K approximately 10(5) vs approximately 10(7) M(-)(1)) but does not alter the binding stoichiometry [12 Fe(II)/dodecamer]. In the H31G-H43G and H31G-H43G-D58A variants, binding of Fe(II) does not take place with measurable affinity. Oxidation of protein-bound Fe(II) increases the binding stoichiometry to 24 Fe(III)/dodecamer. However, the extent of fluorescence quenching upon Fe(III) binding decreases, and the end point near 24 Fe(III)/dodecamer becomes less distinct with increase in the number of mutated residues. In the presence of dioxygen, the mutations have little or no effect on the kinetics of iron uptake and in the formation of micelles inside the protein shell. In contrast, in the presence of hydrogen peroxide, with increase in the number of substitutions the rate of iron oxidation and the capacity to inhibit Fenton chemistry, thereby protecting DNA from oxidative damage, appear increasingly compromised, a further indication of the role of ferroxidation in conferring peroxide tolerance to the bacterium.     

Impaired cytolytic activity in calreticulin-deficient CTLs

Calreticulin is an endoplasmic reticulum-resident chaperone that is stored in the cytotoxic granules of CTLs and NK cells and is released with granzymes and perforin upon recognition of target cells. To investigate the role of calreticulin in CTL-mediated killing, we generated CTL lines from crt(+/+) and crt(-/-) mice expressing a constitutively active form of calcineurin in the heart. Crt(-/-) CTLs showed reduced cytotoxic activity toward allogeneic target cells despite normal production, intracellular localization, and activity of granzymes and despite perforin overexpression. Comparable or higher amounts of granzymes were degranulated by crt(-/-) cells in response to immobilized anti-CD3 Abs, indicating that calreticulin is dispensable for the signal transduction that leads to granule exocytosis. The ability to form conjugates with target cells was affected in the crt(-/-) CTLs, explaining the observed reduction in cytotoxicity. Conjugate formation and cytotoxicity were completely restored by treatments that facilitate recognition and contact with target cells, a prerequisite for degranulation and killing. Therefore, we conclude that calreticulin is dispensable for the cytolytic activity of granzymes and perforin, but it is required for efficient CTL-target cell interaction and for the formation of the death synapse.     

Solution structure and backbone dynamics of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin mutant: a molecular analysis of its reduced thermal stability

No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature.     

The antibiotic polymyxin B modulates P2X7 receptor function

The natural peptide polymyxin B (PMB) is a well-known and potent antibiotic that binds and neutralizes bacterial endotoxin (LPS), thus preventing its noxious effects among LPS-mediated endotoxin shock in animal models. We have investigated the effect of PMB on responses mediated by the P2X(7)R in HEK293 and K562 cells transfected with P2X(7) cDNA and in mouse and human macrophages. In addition, in view of the potential exploitation of P2X(7)-directed agonists in antitumor therapy, we also investigated the effect of PMB in B lymphocytes from patients affected by chronic lymphocytic leukemia. PMB, at an optimal concentration dependent on the given cell type, greatly potentiated the effect of nucleotide-mediated P2X(7) stimulation. In particular, ATP-mediated Ca(2+) influx, plasma membrane permeabilization, and cytotoxicity were enhanced to an extent that, in the presence of PMB, cells were killed by otherwise ineffective nucleotide concentrations. The synergistic effect due to the combined application of ATP and PMB was prevented by incubation with the irreversible P2X blocker oxidized ATP (oATP), but not with the reversible antagonist 1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phenilpiperazine (KN-62). Cells lacking P2X(7) were fully insensitive to the combined stimulation with PMB and ATP. Furthermore, PMB at the concentrations used had no untoward effects on cell viability. These results point to PMB as a useful tool for the modulation of P2X(7)R function and suggest that care should be used in the evaluation of ATP-stimulated immune cell responses in the presence of PMB as they may not solely be affected by removal of contaminating LPS.