Bioclimatic analysis in a region of southern Italy (Calabria)

In this study, an analysis of precipitation and temperature data has been performed over 67 series observed in a region of southern Italy (Calabria). At first, to detect possible trends in the time series, an analysis was performed with the Mann–Kendall non-parametric test applied at monthly and seasonal scale. An additional investigation, useful for checking the climate change effects on vegetation, has also been included analysing bioclimatic indicators. In particular, Emberger, Rivas-Martinez and De Martonne indices were calculated by using monthly temperature and precipitation data in the period 1916–2010. The spatial pattern of the indices has been evaluated and, in order to link the vegetation and the indices, different indices maps have been intersected with the land cover data, given by the Corine Land Cover map. Moreover, the temporal evolution of the indices and of the vegetation has been analysed. Results suggest that climate change may be responsible for the forest cover change, but, given also the good relationship between the various types of bioclimate and forest formations, human activities must be considered.     

Binding of hirudin to human alpha, beta and gamma-thrombin. A comparative kinetic and thermodynamic study.

Thermodynamic parameters for the binding of hirudin to human alpha, beta and gamma-thrombin have been determined between pH 5.0 and 9.0, and from 10 degrees C to 40 degrees C; kinetic data for the association and dissociation of the proteinase-inhibitor complex were obtained at pH 7.5 and 21 degrees C. These results have been analysed in parallel with the inhibitor-binding properties of human alpha, beta and gamma-thrombin for the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI). For the purpose of an homogeneous comparison, values of the apparent association equilibrium constant for BPTI binding to human gamma-thrombin have been determined between pH 5.0 and 9.0, at 21 degrees C. The different binding behaviour of hirudin and BPTI with respect to human alpha, beta and gamma-thrombin has been related to the inferred stereochemistry of the proteinase-inhibitor contact regions. In particular, whereas the beta and gamma-loops play an appreciable role in the stabilization of the enzyme-hirudin complexes, they contribute to impairment of the adduct formation for the proteinase/BPTI system.     

Anticooperative ligand binding properties of recombinant ferric Vitreoscilla homodimeric hemoglobin: a thermodynamic, kinetic and X-ray crystallographic study.

Thermodynamics and kinetics for cyanide, azide, thiocyanate and imidazole binding to recombinant ferric Vitreoscilla sp. homodimeric hemoglobin (Vitreoscilla Hb) have been determined at pH 6.4 and 7.0, and 20.0 degrees C, in solution and in the crystalline state. Moreover, the three-dimensional structures of the diligated thiocyanate and imidazole derivatives of recombinant ferric Vitreoscilla Hb have been determined by X-ray crystallography at 1.8 A (Rfactor=19.9%) and 2.1 A (Rfactor=23.8%) resolution, respectively. Ferric Vitreoscilla Hb displays an anticooperative ligand binding behaviour in solution. This very unusual feature can only be accounted for by assuming ligand-linked conformational changes in the monoligated species, which lead to the observed 300-fold decrease in the affinity of cyanide, azide, thiocyanate and imidazole for the monoligated ferric Vitreoscilla Hb with respect to that of the fully unligated homodimer. In the crystalline state, thermodynamics for azide and imidazole binding to ferric Vitreoscilla Hb may be described as a simple process with an overall ligand affinity for the homodimer corresponding to that for diligation in solution. These data suggest that the ligand-free homodimer, observed in the crystalline state, is constrained in a low affinity conformation whose ligand binding properties closely resemble those of the monoligated species in solution. From the kinetic viewpoint, anticooperativity is reflected by the 300-fold decrease of the second-order rate constant for cyanide and imidazole binding to the monoligated ferric Vitreoscilla Hb with respect to that for ligand association to the ligand-free homodimer in solution. On the other hand, values of the first-order rate constant for cyanide and imidazole dissociation from the diligated and monoligated derivatives of ferric Vitreoscilla Hb in solution are closely similar. As a whole, ligand binding and structural properties of ferric Vitreoscilla Hb appear to be unique among all Hbs investigated to date.     

Peroxynitrite detoxification by ferryl Mycobacterium leprae truncated hemoglobin O

During infection, Mycobacterium leprae is faced with the host macrophagic environment limiting the growth of the bacilli. However, (pseudo-)enzymatic detoxification systems, including truncated hemoglobin O (Ml-trHbO), could allow this mycobacterium to persist in vivo. Here, kinetics of peroxynitrite (ONOOH/ONOO⁻) detoxification by ferryl Ml-trHbO (Ml-trHbOFe(IV)O), obtained by treatment with H₂O₂, is reported. Values of the second-order rate constant for peroxynitrite detoxification by Ml-trHbOFe(IV)O (i.e., of Ml-trHbOFe(III) formation; k_on), at pH 7.2 and 22.0 °C, are 1.5 × 10⁴ M⁻¹ s⁻¹, and 2.2 × 10⁴ M⁻¹ s⁻¹, in the absence of and presence of physiological levels of CO₂ (∼1.2 × 10⁻³ M), respectively. Values of k_on increase on decreasing pH with a pK_a value of 6.7, this suggests that ONOOH reacts preferentially with Ml-trHbOFe(IV)O. In turn, peroxynitrite acts as an antioxidant of Ml-trHbOFe(IV)O, which could be responsible for the oxidative damage of the mycobacterium. As a whole, Ml-trHbO can undertake within the same cycle H₂O₂ and peroxynitrite detoxification.     

Fluctuations and the Rate-Limiting Step of Peptide-Induced Membrane Leakage

Peptide-induced vesicle leakage is a common experimental test for the membrane-perturbing activity of antimicrobial peptides. The leakage kinetics is usually very slow, requiring minutes to hours for complete release of vesicle contents, and exhibits a biphasic behavior. We report here that, in the case of the peptaibol trichogin GA IV, all processes involved in peptide-membrane interaction, such as peptide-membrane association, peptide aggregation, and peptide translocation, take place on a timescale much shorter than the leakage kinetics. On the basis of these findings, we propose a stochastic model in which the leakage kinetics is determined by the discrete nature of a vesicle suspension: peptides are continuously exchanging among vesicles, producing significant fluctuations over time in the number of peptide molecules bound to each vesicle, and in the formation of pores. According to this model, the fast initial leakage is caused by vesicles that contain at least one pore after the peptides are randomly distributed among the liposomes, whereas the slower release is associated with the time needed to occasionally reach in an intact vesicle the critical number of bound peptides necessary for pore formation. Fluctuations due to peptide exchange among vesicles therefore represent the rate-limiting step of such a slow mechanism.     

Effects of a natural extract from Mangifera indica L, and its active compound, mangiferin, on energy state and lipid peroxidation of red blood cells

Following oxidative stress, modifications of several biologically important macromolecules have been demonstrated. In this study we investigated the effect of a natural extract from Mangifera indica L (Vimang), its main ingredient mangiferin and epigallocatechin gallate (EGCG) on energy metabolism, energy state and malondialdehyde (MDA) production in a red blood cell system. Analysis of MDA, high energy phosphates and ascorbate was carried out by high performance liquid chromatography (HPLC). Under the experimental conditions, concentrations of MDA and ATP catabolites were affected in a dose-dependent way by H2O2. Incubation with Vimang (0.1, 1, 10, 50 and 100 microg/mL), mangiferin (1, 10, 100 microg/mL) and EGCG (0.01, 0.1, 1, 10 microM) significantly enhances erythrocyte resistance to H2O2-induced reactive oxygen species production. In particular, we demonstrate the protective activity of these compounds on ATP, GTP and total nucleotides (NT) depletion after H2O2-induced damage and a reduction of NAD and ADP, which both increase because of the energy consumption following H2O2 addition. Energy charge potential, decreased in H2O2-treated erythrocytes, was also restored in a dose-dependent way by these substances. Their protective effects might be related to the strong free radical scavenging ability described for polyphenols.     

Allogeneic mesoangioblasts give rise to alpha-sarcoglycan expressing fibers when transplanted into dystrophic mice

Cell therapy for muscular dystrophy involves transplantation of either genetically modified autologous cells or normal donor cells that will be rejected unless the host is adequately immune suppressed. The extent of the immune response appears to be mitigated in this case of stem cells, by immune-suppressive and tolerogenic molecules that they release. We previously reported significant morphological and functional amelioration of a mouse model of limb-girdle muscular dystrophy by transplantation of mesoangioblasts. These are vessel-associated stem cells that can be propagated in vitro and differentiate into several types of mesoderm including skeletal muscle. In these experiments, both donor cells and host were syngeneic (C57Bl/6J) and thus possible immune reaction to the donor cells could not be appreciated. To address this question, we transplanted H2-mismatched mesoangioblasts (BalbC) in the same dystrophic mice, and in addition, we treated the host with different pharmacological drugs (rapamycin, IL-10 or both). The results showed that donor cells give rise to fibers that express the mutated gene product (alpha-sarcoglycan) even in the absence of immune suppression; however, the combined action of rapamycin and IL-10 increases the number of alpha-sarcoglycan expressing fibers while reducing the levels of inflammatory cytokines. These results indicate that transplantation of mesoangioblasts into immunologically unrelated host leads to long-term survival of donor cells and this may be further enhanced by appropriate protocols of immune modulation, thus setting the stage for experimentation in large animals and in patients.