Molecular properties of lysozyme-microbubbles: towards the protein and nucleic acid delivery

Microbubbles (MBs) have specific acoustic properties that make them useful as contrast agents in ultrasound imaging. The use of the MBs in clinical practice led to the development of more sensitive imaging techniques both in cardiology and radiology. Protein-MBs are typically obtained by dispersing a gas phase in the protein solution and the protein deposited/cross-linked on the gas-liquid interface stabilizes the gas core. Innovative applications of protein-MBs prompt the investigation on the properties of MBs obtained using different proteins that are able to confer them specific properties and functionality. Recently, we have synthesized stable air-filled lysozyme-MBs (LysMBs) using high-intensity ultrasound-induced emulsification of a partly reduced lysozyme in aqueous solutions. The stability of LysMBs suspension allows for post-synthetic modification of MBs surface. In the present work, the protein folded state and the biodegradability property of LysMBs were investigated by limited proteolysis. Moreover, LysMBs were coated and functionalized with a number of biomacromolecules (proteins, polysaccharides, nucleic acids). Remarkably, LysMBs show a high DNA-binding ability and protective effects of the nucleic acids from nucleases and, further, the ability to transform the bacteria cells. These results highlight on the possibility of using LysMBs for delivery of proteins and nucleic acids in prophylactic and therapeutic applications.     

Assessing influence in biofuel production and ecosystem services when environmental changes affect plant–pest relationships

Climate change is currently affecting both biodiversity and human activities; land use change and greenhouse gas emissions are the main drivers. Many agricultural services are affected by the change, which in turn reflects on the basic provisioning services, which supply food, fibre and biofuels. Biofuels are getting increasing interest because of their sustainability potential. Jatropha curcas gained popularity as a biodiesel crop, due to its ease of cultivation even in harsh environmental conditions. Notwithstanding its high economic importance, few studies are available about its co‐occurrence with pests of the genus Aphthona in sub‐Saharan Africa, where these insects feed on J. curcas, leading to relevant economic losses. Using ecological niche modelling and GIS post‐modelling analyses, we infer the current and future suitable territories for both these taxa, delineating areas where J. curcas cultivations may occur without suffering the presence of Aphthona, in the context of future climate and land use changing. We introduce an area‐normalized index, the ‘Potential‐Actual Cultivation Index’, to better depict the ratio between the suitable areas shared both by the crop and its pest, and the number of actual cultivations, in a target country. Moreover, we find high economic losses (~?50%) both in terms of carbon sequestration and in biodiesel production when J. curcas co‐occur with the Aphthona cookei species group.     

Analysis of grape berry cell wall proteome: a comparative evaluation of extraction methods

Different methods were tested for the extraction of proteins from the cell wall-enriched fraction (CWEf) obtained from a sample formed by skin and seeds of ripe berries of Vitis vinifera L. cv. Cabernet Sauvignon. The CWEf was isolated using a disruptive approach that involves tissue homogenization and precipitation by centrifugation. To extract proteins, the CWEf was treated with CaCl(2) and LiCl in two successive steps or, alternatively, with phenol. The efficiency of the protocols was evaluated by measuring protein yield and by analyzing two-dimensional gel electrophoresis (2-DE) gels for the highest detectable spot number and the greatest spot resolution. The phenol method was also adopted for the extraction of proteins from the cytosolic fraction (CYf). The comparison of 2-DE reference maps of protein extracts from CWEf and CYf indicated the presence of both common traits and unique characteristics. To survey this aspect some spots detected in both fractions or present in only one fraction were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Of the 47 spots identified, some were found to be cell wall proteins, while others were proteins not traditionally considered as localized in the apoplastic space. The data presented here provide initial information regarding the apoplastic proteome of grape berry tissues, but also raise the issue of the technical problems that characterize the isolation of cell wall proteins from these very hardy tissues.     

Neuroglobin: enzymatic reduction and oxygen affinity

Neuroglobin (Ngb) is a hexacoordinate globin expressed in the nervous system of vertebrates, involved in neuroprotection. O₂ equilibrium measurements on mouse Ngb yielded significantly different P₅₀ values, ranging from ∼2 torr to ∼10 torr. By a kinetic approach minimizing the effects of protein autoxidation, we measured P₅₀ = 2.2 torr at 20 °C. As predicted from the structure, O₂ binds to the Y44D Ngb mutant more quickly (k = 2.2 s⁻¹ vs 0.15 s⁻¹) and with slightly higher affinity (P₅₀ = 1.3 torr) than wild-type. In addition, we introduced a novel reduction protocol for metNgb based on NADH:flavorubredoxin oxidoreductase (FlRd-red) from Escherichia coli, a candidate for the Ngb reducing activity recently identified in E. coli extracts. Interestingly, E. coli FlRd-red shares sequence similarity with the FAD-binding domain of the human apoptosis-inducing factor, a finding which may have unexpected significance with reference to the mechanism of neuroprotection by Ngb.     

Chemodiversity of the Essential Oil from Leaves of Abies nebrodensis (Lojac.) Mattei

Abies nebrodensis (Lojac .) Mattei (Pinaceae) is a species occurring in a very small population only in a restricted area of Sicily. Its taxonomic classification as different species has been object of discussion. In this work the chemical composition of the essential oil from the leaves is presented for the first time and compared to the essential oils from other euroasiatic species reported in literature. Peculiar characteristics of the essential oil of A. nebrodensis are highlighted.     

Protein phosphatase 2A subunit PR70 interacts with pRb and mediates its dephosphorylation

The retinoblastoma tumor suppressor protein (pRb) regulates cell proliferation and differentiation via phosphorylation-sensitive interactions with specific targets. While the role of cyclin/cyclin-dependent kinase complexes in the modulation of pRb phosphorylation has been extensively studied, relatively little is known about the molecular mechanisms regulating phosphate removal by phosphatases. Protein phosphatase 2A (PP2A) is constituted by a core dimer bearing catalytic activity and one variable B regulatory subunit conferring target specificity and subcellular localization. We previously demonstrated that PP2A core dimer binds pRb and dephosphorylates pRb upon oxidative stress. In the present study, we identified a specific PP2A-B subunit, PR70, that was associated with pRb both in vitro and in vivo. PR70 overexpression caused pRb dephosphorylation; conversely, PR70 knockdown prevented both pRb dephosphorylation and DNA synthesis inhibition induced by oxidative stress. Moreover, we found that intracellular Ca²⁺ mobilization was necessary and sufficient to trigger pRb dephosphorylation and PP2A phosphatase activity of PR70 was Ca²⁺ induced. These data underline the importance of PR70-Ca²⁺ interaction in the signal transduction mechanisms triggered by redox imbalance and leading to pRb dephosphorylation.     

Degradation of aromatic hydrocarbons by white-rot fungi in a historically contaminated soil

Phanerochaete chrysosporium NRRL 6361 and Pleurotus pulmonarius CBS 664.97 were tested for their ability to grow under nonsterile conditions and to degrade various aromatic hydrocarbons in an aged contaminated soil that also contained high concentrations of heavy metals. After 24 days fungal incubation, carbon-CO2 liberated, an indicator of microbial activity, reached a plateau. At the end of the incubation time (30 days), fungal colonization was clearly visible and was confirmed by ergosterol and cell organic carbon determinations. In spite of unfavorable pH (around 7.4) and the presence of heavy metals, both fungi produced Mn-peroxidase activity. In contrast, laccase and aryl-alcohol oxidase were detected only in the soil treated with P. pulmonarius CBS 664.97 and lignin-peroxidase in that with P. chrysosporium NRRL 6361. No lignin-modifying enzyme activities were present in non-inoculated soil incubated for 30 days (control microcosm). Regardless of the fungus employed, a total removal of naphtalene, tetrachlorobenzene, and dichloroaniline isomers, diphenylether and N-phenyl-1-naphtalenamine, was observed. Significant release of chloride ions was also observed in fungal-treated soil, in comparison with that recorded in the control microcosm. Both fungi led to a significant decrease in soil toxicity, as assessed using two different soil contact assays, including the Lepidium sativum L. germination test and the Collembola mortality test.     

Characterization of the major allergens purified from the venom of the paper wasp Polistes gallicus

Allergic reactions to vespid stings are one of the major causes of IgE-mediated anaphylaxis. Vespa and Vespula venoms are closely related; Polistes venom is more distantly related and its allergens are less well studied. There is limited cross-reactivity between Polistes and the other vespid venoms because of differences in the epitopes on the allergen molecules.In this study, the major allergens of Polistes gallicus are isolated and characterized. P. gallicus venom contains four major allergens: phospholipase, antigen 5 (Ag5), hyaluronidase and protease that were characterized by mass spectrometry and specific binding to IgE. The complete amino acid sequence of Ag5 and the sequence of the N-terminal region of phospholipase were also determined. The alignment of Ag5 from P. gallicus (European species) and Polistes annularis (American species) shows an 85% identity that increases to 98% within the same subgenus. This could suggest the presence of specific epitopes on Ag5 molecule being the variations on the superficial loops. The features of the P. gallicus allergens could explain the partial cross-reactivity found between the American and European Polistes venoms, and suggest that the use of European Polistes venoms would improve the diagnostic specificity and the therapy of European patients and of North American patients sensitized by European Polistes.     

The three-dimensional structures of the Mycobacterium tuberculosis dihydrodipicolinate reductase-NADH-2,6-PDC and -NADPH-2,6-PDC complexes. Structural and mutagenic analysis of relaxed nucleotide specificity

Dihydrodipicolinate reductase (DHPR) catalyzes the reduced pyridine nucleotide-dependent reduction of the alpha,beta-unsaturated cyclic imine, dihydrodipicolinate, to generate tetrahydrodipicolinate. This enzyme catalyzes the second step in the bacterial biosynthetic pathway that generates meso-diaminopimelate, a component of bacterial cell walls, and the amino acid L-lysine. The Mycobacterium tuberculosis dapB-encoded DHPR has been cloned, expressed, purified, and crystallized in two ternary complexes with NADH or NADPH and the inhibitor 2,6-pyridinedicarboxylate (2,6-PDC). The structures have been solved using molecular replacement strategies, and the DHPR-NADH-2,6-PDC and DHPR-NADPH-2,6-PDC complexes have been refined against data to 2.3 and 2.5 A, respectively. The M. tuberculosis DHPR is a tetramer of identical subunits, with each subunit composed of two domains connected by two flexible hinge regions. The N-terminal domain binds pyridine nucleotide, while the C-terminal domain is involved in both tetramer formation and substrate/inhibitor binding. The M. tuberculosis DHPR uses NADH and NADPH with nearly equal efficiency based on V/K values. To probe the nature of this substrate specificity, we have generated two mutants, K9A and K11A, residues that are close to the 2'-phosphate of NADPH. These two mutants exhibit decreased specificity for NADPH by factors of 6- and 30-fold, respectively, but the K11A mutant exhibits 270% of WT activity using NADH. The highly conserved structure of the nucleotide fold may permit other enzyme's nucleotide specificity to be altered using similar mutagenic strategies.     

Indigenous Vibrio cholerae strains from a non-endemic region are pathogenic

Of the 200+ serogroups of Vibrio cholerae, only O1 or O139 strains are reported to cause cholera, and mostly in endemic regions. Cholera outbreaks elsewhere are considered to be via importation of pathogenic strains. Using established animal models, we show that diverse V. cholerae strains indigenous to a non-endemic environment (Sydney, Australia), including non-O1/O139 serogroup strains, are able to both colonize the intestine and result in fluid accumulation despite lacking virulence factors believed to be important. Most strains lacked the type three secretion system considered a mediator of diarrhoea in non-O1/O13 V. cholerae. Multi-locus sequence typing (MLST) showed that the Sydney isolates did not form a single clade and were distinct from O1/O139 toxigenic strains. There was no correlation between genetic relatedness and the profile of virulence-associated factors. Current analyses of diseases mediated by V. cholerae focus on endemic regions, with only those strains that possess particular virulence factors considered pathogenic. Our data suggest that factors other than those previously well described are of potential importance in influencing disease outbreaks.