Spinodal lines and Flory-Huggins free-energies for solutions of human hemoglobins HbS and HbA.

Gelation of deoxygenated solutions of sickle-cell human Hemoglobin (HbS) is of high theoretical interest and it has serious pathological consequences. For this reason HbS is probably the most studied protein capable of self-organization. This notwithstanding, the location in the T, c plane of the region of thermodynamic instability of solutions of deoxy-HbS (as bounded by the spinodal line and as distinct from the gelation region) has remained unknown, along with related values of Flory-Huggins enthalpies and entropies. In the present work this information is derived from experiments for the two cases of (deoxy) HbS and of human adult hemoglobin (HbA). Experiments also show critical exponents having mean-field values, which validates a Flory-Huggins approach. Altogether, the present work offers a quantitative understanding of the thermodynamic effects of the genetic HbA----HbS mutation and it opens the way to similar quantitative evaluations of contributions of pH, salts, cosolutes, and single peptides (even for nongelling hemoglobins), and of potential therapeutic strategies.     

Secretome Analysis of Hypoxia‐Induced 3T3‐L1 Adipocytes Uncovers Novel Proteins Potentially Involved in Obesity

In the obese state, as adipose tissue expands, adipocytes become hypoxic and dysfunctional, leading to changes in the pattern of adipocyte‐secreted proteins. To better understand the role of hypoxia in the mechanisms linked to obesity, we comparatively analyzed the secretome of murine differentiated 3T3‐L1 adipocytes exposed to normoxia or hypoxia for 24 h. Proteins secreted into the culture media were precipitated by trichloroacetic acid and then digested with trypsin. The peptides were labeled with dimethyl labeling and analyzed by reversed phase nanoscale liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. From a total of 1508 identified proteins, 109 were differentially regulated, of which 108 were genuinely secreted. Factors significantly downregulated in hypoxic conditions included adiponectin, a known adipokine implicated in metabolic processes, as well as thrombospondin‐1 and ‐2, and matrix metalloproteinase‐11, all multifunctional proteins involved in extracellular matrix (ECM) homeostasis. Findings were validated by Western blot analysis. Expression studies of the relative genes were performed in parallel experiments in vitro, in differentiated 3T3‐L1 adipocytes, and in vivo, in fat tissues from obese versus lean mice. Our observations are compatible with the concept that hypoxia may be an early trigger for both adipose cell dysfunction and ECM remodeling.     

Collective properties of hydration: long range and specificity of hydrophobic interactions.

We report results of molecular dynamics (MD) simulations of composite model solutes in explicit molecular water solvent, eliciting novel aspects of the recently demonstrated, strong many-body character of hydration. Our solutes consist of identical apolar (hydrophobic) elements in fixed configurations. Results show that the many-body character of PMF is sufficiently strong to cause 1) a remarkable extension of the range of hydrophobic interactions between pairs of solute elements, up to distances large enough to rule out pairwise interactions of any type, and 2) a SIF that drives one of the hydrophobic solute elements toward the solvent rather than away from it. These findings complement recent data concerning SIFs on a protein at single-residue resolution and on model systems. They illustrate new important consequences of the collective character of hydration and of PMF and reveal new aspects of hydrophobic interactions and, in general, of SIFs. Their relevance to protein recognition, conformation, function, and folding and to the observed slight yet significant nonadditivity of functional effects of distant point mutations in proteins is discussed. These results point out the functional role of the configurational and dynamical states (and related statistical weights) corresponding to the complex configurational energy landscape of the two interacting systems: biomolecule + water.     

Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: the case of Minibody.

A strategy that combines limited proteolysis experiments and mass spectrometric analysis of the fragments generated has been developed to probe protease-accessible sites on the protein surface. This integrated approach has been employed to investigate the tertiary structure of the Minibody, a de novo designed 64-residue protein consisting of a beta-sheet scaffold based on the heavy-chain variable-domain structure of a mouse immunoglobulin and containing two segments corresponding to the hypervariable H1 and H2 regions. The low solubility of the protein prevented a detailed characterization by NMR and/or X-ray. Different proteases were used under strictly controlled conditions and the cleavage sites were mapped onto the anticipated Minibody model, leading to the identification of the most exposed regions. A single-residue mutant was constructed and characterized, following the same procedure, showing a slightly higher correspondence with the predicted model. This strategy can be used to effectively supplement NMR and X-ray investigations of protein tertiary structure, where these procedures cannot provide definitive data, or to verify and refine protein models.     

HIV-1 A1 Subtype Epidemic in Italy Originated from Africa and Eastern Europe and Shows a High Frequency of Transmission Chains Involving Intravenous Drug Users

Background

Subtype A accounts for only 12% of HIV-1 infections worldwide but predominates in Russia and Former Soviet Union countries of Eastern Europe. After an early propagation via heterosexual contacts, this variant spread explosively among intravenous drug users. A distinct A1 variant predominates in Greece and Albania, which penetrated directly from Africa. Clade A1 accounts for 12.5% of non-B subtypes in Italy, being the most frequent after F1 subtype.

Aim

Aim of this study was to investigate the circulation of A1 subtype in Italy and trace its origin and diffusion through phylogenetic and phylodynamic approaches.

Results

The phylogenetic analysis of 113 A1 pol sequences included in the Italian ARCA database, indicated that 71 patients (62.8%) clustered within 5 clades. A higher probability to be detected in clusters was found for patients from Eastern Europe and Italy (88.9% and 60.4%, respectively) compared to those from Africa (20%) (p < .001). Higher proportions of clustering sequences were found in intravenous drug users with respect to heterosexuals (85.7% vs. 59.3%, p = .056) and in women with respect to men (81.4% vs. 53.2%, p < .006). Subtype A1 dated phylogeny indicated an East African origin around 1961. Phylogeographical reconstruction highlighted 3 significant groups. One involved East European and some Italian variants, the second encompassed some Italian and African strains, the latter included the majority of viruses carried by African and Italian subjects and all viral sequences from Albania and Greece.

Conclusions

Subtype A1 originated in Central Africa and spread among East European countries in 1982. It entered Italy through three introduction events: directly from East Africa, from Albania and Greece, and from the area encompassing Moldavia and Ukraine. As in previously documented A1 epidemics of East European countries, HIV-1 A1 subtype spread in Italy in part through intravenous drug users. However, Eastern European women contributed to the penetration of such variant, probably through sex work.     

Effect of some organic cosolvents on the reaction of hemoglobin with oxygen

We studied the effects of some organic cosolvents (monohydric alcohols and amides) on the reaction of hemoglobin with oxygen. We present evidence showing that our data can be analyzed within the framework of the Monod-Wyman-Changeux model and that the main effect of cosolvents is to alter the T ? R conformational equilibrium of hemoglobin, without significantly affecting the intrinsic oxygen dissociation constants. Following a previously described phenomenological approach, the overall effects have been separated into effects related to the variation of the bulk dielectric constant of the solvent and effects not related to the variation of this constant.