Binding of small phosphorylated chromatin peptides to DNA

Low-molecular-weight peptides involved in gene expression and cell growth have been isolated from DNA preparation from eukaryotic cells. After phosphorylation with protein kinase CKII (pCKII) these peptides are able to bind to DNA in presence of divalent cations and salt/ethanol. This finding may explain the mechanism by which the peptides exert their activity.     

Lower extremity muscle size and strength and aerobic capacity decrease with caloric restriction but not with exercise-induced weight loss.

Caloric restriction (CR) results in fat loss; however, it may also result in loss of muscle and thereby reduce strength and aerobic capacity (VO2 max). These effects may not occur with exercise-induced weight loss (EX) because of the anabolic effects of exercise on heart and skeletal muscle. We tested the hypothesis that CR reduces muscle size and strength and VO2 max, whereas EX preserves or improves these parameters. Healthy 50- to 60-yr-old men and women (body mass index of 23.5-29.9 kg/m2) were studied before and after 12 mo of weight loss by CR (n = 18) or EX (n = 16). Lean mass was assessed by dual-energy X-ray absorptiometry, thigh muscle volume by MRI, isometric and isokinetic knee flexor strength by dynamometry, and treadmill VO2 max by indirect calorimetry. Both interventions caused significant decreases in body weight (CR: -10.7 +/- 1.4%, EX: -9.5 +/- 1.5%) and lean mass (CR: -3.5 +/- 0.7%, EX: -2.2 +/- 0.8%), with no significant differences between groups. Significant decreases in thigh muscle volume (-6.9 +/- 0.8%) and composite knee flexion strength (-7.2 +/- 3%) occurred in the CR group only. Absolute VO2 max decreased significantly in the CR group (-6.8 +/- 2.3%), whereas the EX group had significant increases in both absolute (+15.5 +/- 2.4%) and relative (+28.3 +/- 3.0%) VO2 max. These data provide evidence that muscle mass and absolute physical work capacity decrease in response to 12 mo of CR but not in response to a similar weight loss induced by exercise. These findings suggest that, during EX, the body adapts to maintain or even enhance physical performance capacity.     

Effects of the inoculation of cyanobacteria on the microstructure and the structural stability of a tropical soil

Cyanobacteria are widespread photosynthetic microorganisms among which some are able to fix atmospheric nitrogen. We investigated the impact of indigenous cyanobacteria strains (Nostoc) inoculation on physical characteristics of poorly aggregated soils from Guquka (Eastern Cape, South Africa). The soil aggregates (3–5 mm) were arranged into a layer of 10–20 mm thick, and sprayed with cyanobacteria solution. Subsequently the inoculated and un-inoculated samples were incubated (30°C, 80% humidity, continuous illumination at 100 μmol m⁻² s⁻¹). Their micromorphological characteristics and aggregate stability were investigated, after 1, 2, 3, 4 and 6 weeks of incubation, by using high resolution Cryo-SEM and aggregate breakdown tests. Micromorphological investigations revealed that the surface of un-inoculated samples remained uncovered, while the inoculated samples were partially covered by cyanobacteria material after one week of incubation. A dense superficial network of cyanobacterial filaments and extracellular polymer secretions (EPS) covered their surface after 4 and 6 weeks of incubation. Organo-mineral aggregates comprising cyanobacterial filaments and EPS were observed after 6 weeks of incubation. The results of aggregate breakdown tests showed no significant difference between un-inoculated samples after 1, 2, 3, 4 or 6 weeks, while they revealed improvement of aggregate stability for inoculated samples. The improvement of aggregate stability appeared in a short while following inoculation and increased gradually with time and cyanobacteria growth. The increase in aggregate stability is likely related to the changes induced in micromorphological characteristics by cyanobacterial filaments and EPS. It reflects the effect of coating, enmeshment, binding and gluing of aggregates and isolated mineral particles by cyanobacteria material. Our study presents new data demonstrating the improvement of soil physical quality in a few weeks after cyanobacteria inoculation. The interaction of the inocula and other biotic components is worthy of study before field application of cyanobacteria.     

MG-63 human osteosarcoma cells grown in monolayer and as three-dimensional tumor spheroids present a different metabolic profile: a (1)H NMR study

High resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy was used to determine if the same cell line (MG-63 human osteosarcoma cells) grown in monolayer or as small (about 50-80 microm in diameter), three-dimensional tumor spheroids with no hypoxic center has different metabolic characteristics. Consequently, the (1)H NMR spectra were obtained from both types of cultures and then compared. The results indicate that the type of cellular spatial array determines specific changes in MG-63 cells. In particular, small but significant differences in lactate and alanine indicating a perturbation in energy metabolism were observed in the two cell models. In addition, although variations in CH(2) and CH(3) groups were also seen, it is not possible at this time to establish if lipid metabolism is truly different in cells and spheroids.     

Stability and structure of telomeric DNA sequences forming quadruplexes containing four G-tetrads with different topological arrangements

Telomeres are DNA-protein structures at the ends of eukaryotic chromosomes, the DNA of which comprise noncoding repeats of guanine-rich sequences. Telomeric DNA plays a fundamental role in protecting the cell from recombination and degradation. Telomeric sequences can form quadruplex structures stabilized by guanine quartets. These structures can be constructed from one, two, or four oligonucleotidic strands. Here, we report the thermodynamic characterization of the stability, analyzed by differential scanning calorimetry, of three DNA quadruplexes of different molecularity, all containing four G-tetrads. The conformational properties of these quadruple helices were studied by circular dichroism. The investigated oligomers form well-defined G-quadruplex structures in the presence of sodium ions. Two have the truncated telomeric sequence from Oxytricha, d(TGGGGT) and d(GGGGTTTTGGGG), which form a tetramolecular and bimolecular quadruplex, respectively. The third sequence, d(GGGGTTGGGGTGTGGGGTTGGGG) was designed to form a unimolecular quadruplex. The thermodynamic parameters of these quadruplexes have been determined. The tetramolecular structure is thermodynamically more stable than the bimolecular one, which, in turn, is more stable than the unimolecular one. The experimental data were discussed in light of the molecular-modeling study.     

The crystal structure of Sulfolobus solfataricus elongation factor 1alpha in complex with magnesium and GDP

Recent studies have shown that elongation factors extracted from archaea/eukarya and from eubacteria exhibit different structural and functional properties. Along this line, it has been demonstrated that, in contrast to EF-Tu, Sulfolobus solfataricus EF-1alpha in complex with GDP (SsEF-1alpha.GDP) does not bind Mg(2+), when the ion is present in the crystallization medium at moderate concentration (5 mM). To further investigate the role that magnesium plays in the exchange process of EF-1alpha and to check the ability of SsEF-1alpha.GDP to bind the ion, we have determined the crystal structure of SsEF-1alpha.GDP in the presence of a nonphysiological concentration (100 mM) of Mg(2+). The analysis of the coordination of Mg(2+) unveils the structural bases for the marginal role played by the ion in the nucleotide exchange process. Furthermore, nucleotide exchange experiments carried out on a truncated form of SsEF-1alpha, consisting only of the nucleotide binding domain, demonstrate that the low affinity of SsEF-1alpha.GDP for Mg(2+) is due to the local architecture of the active site and does not depend on the presence of the other two domains. Finally, considering the available structures of EF-1alpha, a detailed mechanism for the nucleotide exchange process has been traced. Notably, this mechanism involves residues such as His14, Arg95, Gln131, and Glu134, which are strictly conserved in all archaea and eukarya EF-1alpha sequences hitherto reported.     

Inactivation of parasite cysteine proteinases by the NO-donor 4-(phenylsulfonyl)-3-((2-(dimethylamino)ethyl)thio)-furoxan oxalate

NO-donors block Plasmodium, Trypanosoma, and Leishmania life cycle by inactivating parasite enzymes, e.g., cysteine proteinases. In this study, the inactivation of falcipain, cruzipain, and Leishmania infantum cysteine proteinase by the NO-donor 4-(phenylsulfonyl)-3-((2-(dimethylamino)ethyl)thio)-furoxan oxalate (SNO-102) is reported. SNO-102 inactivates dose- and time-dependently parasite cysteine proteinases; one equivalent of NO, released from SNO-102, inactivates one equivalent of L. infantum cysteine proteinase. With SNO-102 in excess over the parasite cysteine proteinase, the time course of enzyme inhibition corresponds to a pseudo-first-order reaction for more than 90% of its course. The concentration dependence of the pseudo-first-order rate constant is second-order at low SNO-102 concentration but tends to first-order at high NO-donor concentration. This behavior may be explained by a relatively fast pre-equilibrium followed by a limiting pseudo-first order process. Kinetic parameters of L. infantum cysteine proteinase inactivation by SNO-102 are affected by the acidic pK shift of one apparent ionizing group (from pK(unl)=5.8 to pK(lig)=4.7) upon enzyme inhibition. Falcipain, cruzipain and L. infantum cysteine proteinase inactivation is prevented and reversed by dithiothreitol and L-ascorbic acid. Furthermore, the fluorogenic substrate N-alpha-benzyloxycarbonyl-Phe-Arg-(7-amino-4-methylcoumarin) protects parasite cysteine proteinases from inactivation by SNO-102. The absorption spectrum of the inactive S-nitrosylated SNO-102-treated L. infantum cysteine proteinase displays a maximum at about 340 nm. These results indicate that the parasite cysteine proteinase inactivation by SNO-102 occurs via the NO-mediated S-nitrosylation of the Cys25 catalytic residue.     

Solvent-induced ligand dissociation and conformational states of Cellular Retinol-Binding Protein type I

Cellular Retinol-Binding Protein type I (CRBP) exhibits very high affinity for its ligand, bound within a buried cavity completely shielded from the outside medium. Three-dimensional structure and backbone dynamics in aqueous solution at neutral pH, either in the absence or in the presence of retinol, fail to represent the protein in a state capable of ligand uptake and release. The question was asked whether changes in the composition of the outside medium might facilitate ligand dissociation. Acidic aqueous solutions and water-alcohol mixtures were selected, among the best described denaturing solvents, to investigate their effects on the stability of the carrier-ligand complex and the conformational state of the protein upon ligand release. Circular dichroism (CD) and fluorescence spectroscopy were used to probe protein secondary and tertiary structure, compactness and retinol dissociation. While in purely aqueous media retinol dissociation parallels the acid-induced denaturation of the carrier, in water-alcohol mixtures it occurs in a range of co-solvent content lower than that required for protein denaturation. In light of these results, it is suggested that local solvent properties in vivo might modulate protein conformation and flexibility and thus play a fundamental role in the control of retinol exchange between carrier and membrane-bound donors and acceptors.     

Dynamic properties of the N-terminal swapped dimer of ribonuclease A

Bovine pancreatic ribonuclease (RNase A) forms two 3-dimensional domain-swapped dimers with different quaternary structures. One dimer is characterized by the swapping of the C-terminal region (C-Dimer) and presents a rather loose structure. The other dimer (N-Dimer) exhibits a very compact structure with exchange of the N-terminal helix. Here we report the results of a molecular dynamics/essential dynamics (MD/ED) study carried out on the N-Dimer. This investigation, which represents the first MD/ED analysis on a three-dimensional domain-swapped enzyme, provides information on the dynamic properties of the active site residues as well as on the global motions of the dimer subunits. In particular, the analysis of the flexibility of the active site residues agrees well with recent crystallographic and site-directed mutagenesis studies on monomeric RNase A, thus indicating that domain swapping does not affect the dynamics of the active sites. A slight but significant rearrangement of N-Dimer quaternary structure, favored by the formation of additional hydrogen bonds at subunit interface, has been observed during the MD simulation. The analysis of collective movements reveals that each subunit of the dimer retains the functional breathing motion observed for RNase A. Interestingly, the breathing motion of the two subunits is dynamically coupled, as they open and close in phase. These correlated motions indicate the presence of active site intercommunications in this dimer. On these bases, we propose a speculative mechanism that may explain negative cooperativity in systems preserving structural symmetry during the allosteric transitions.