Structural and functional characterization of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Tick defensins may serve as templates for the development of multifunctional peptides. The purpose of this study was to evaluate shorter peptides derived from tick defensin isoform 2 (OsDef2) in terms of their antibacterial, antioxidant, and cytotoxic activities. We compared the structural and functional properties of a synthetic peptide derived from the carboxy‐terminal of the parent peptide (Os) to that of an analogue in which the three cysteine residues were omitted (Os–C). Here, we report that both peptides were bactericidal (MBC values ranging from 0.94–15 µg/ml) to both Gram‐positive and Gram‐negative bacteria, whereas the parent peptide only exhibited Gram‐positive antibacterial activity. The Os peptide was found to be two‐fold more active than Os–C against three of the four tested bacteria but equally active against Staphylococcus aureus. Os showed rapid killing kinetics against both Escherichia coli and Bacillus subtilis, whereas Os–C took longer, suggesting different modes of action. Scanning electron microscopy showed that in contrast to melittin for which blebbing of bacterial surfaces was observed, cells exposed to either peptide appeared flattened and empty. Circular dichroism data indicated that in a membrane‐mimicking environment, the cysteine‐containing peptide has a higher α‐helical content. Both peptides were found to be non‐toxic to mammalian cells. Moreover, the peptides displayed potent antioxidant activity and were 12 times more active than melittin. Multifunctional peptides hold potential for a wide range of clinical applications and further investigation into their mode of antibacterial and antioxidant properties is therefore warranted. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The dual functionality of antimicrobial peptides Os and Os‐C in human leukocytes

Antimicrobial peptides (AMPs), Os and Os‐C, have been identified as multifunctional peptides with antibacterial, antiendotoxin, and anti‐inflammatory properties. For further development of Os and Os‐C as therapeutic peptides, it is essential to evaluate these effects in human mononuclear (MN) and polymorphonuclear (PMN) leukocytes. The cytotoxicity and the effects of both peptides on MN and PMN morphology were determined with the Alamar‐Blue assay and scanning electron microscopy, respectively. The ability of Os and Os‐C to induce reactive oxygen species (ROS) and to protect against 2,2′‐azobis(2‐amidinopropane) dihydrochloride–induced oxidative damage in both cell populations was evaluated using 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA). Using fluorescently labeled peptides, the ability of the peptides to cross the cell membranes of MN and PMN was also evaluated. At the minimum bactericidal concentrations of Os and Os‐C, neither peptide was cytotoxic. Os caused morphological features of toxicity at 100 μM, entered MN cells, and also protected these cells against oxidative damage. Os‐C caused MN and PMN leukocyte activation associated with ROS formation and was unable to penetrate cell membranes, indicating extracellular membrane interactions. This study confirms that both Os and Os‐C at less than 100 μM are not cytotoxic. The MN‐specific uptake of Os identifies it as a cell‐specific cargo‐carrier peptide, with additional anti‐inflammatory properties. In contrast, the ability of Os‐C to activate MN and PMN cells implies that this peptide should be further evaluated as an AMP, which, in addition to its ability to eradicate infection, can further enhance host immunity. These novel characteristics of Os and Os‐C indicate that these AMPs as peptides can be further developed for specific applications.     

Relative Pigment Composition and Remote Sensing Reflectance of Caribbean Shallow-Water Corals

Reef corals typically contain a number of pigments, mostly due to their symbiotic relationship with photosynthetic dinoflagellates. These pigments usually vary in presence and concentration and influence the spectral characteristics of corals. We studied the variations in pigment composition among seven Caribbean shallow-water Scleractinian corals by means of High Performance Liquid Chromatography (HPLC) analysis to further resolve the discrimination of corals. We found a total of 27 different pigments among the coral species, including some alteration products of the main pigments. Additionally, pigments typically found in endolithic algae were also identified. A Principal Components Analysis and a Hierarchical Cluster Analysis showed the separation of coral species based on pigment composition. All the corals were collected under the same physical environmental conditions. This suggests that pigment in the coral’s symbionts might be more genetically-determined than influenced by prevailing physical conditions of the reef. We further investigated the use of remote sensing reflectance (Rrs) as a tool for estimating the total pigment concentration of reef corals. Depending on the coral species, the Rrs and the total symbiont pigment concentration per coral tissue area correlation showed 79.5–98.5% confidence levels demonstrating its use as a non-invasive robust technique to estimate pigment concentration in studies of coral reef biodiversity and health.     

Fungal diversity in Sahara dust: Aspergillus sydowii and other opportunistic pathogens

Aerobiologia - The Caribbean is influenced by Sahara Dust Storms (SDS) every year. SDS can transport a diversity of microorganisms, including potential pathogens of humans, animals, and plants. In...     

Influence of the unusual covalent adduct on the kinetics and formation of radical intermediates in synechocystis catalase peroxidase: a stopped-flow and EPR characterization of the MET275, TYR249, and ARG439 variants

Catalase-peroxidases (KatGs) are heme peroxidases with a catalatic activity comparable to monofunctional catalases. They contain an unusual covalent distal side adduct with the side chains of Trp(122), Tyr(249), and Met(275) (Synechocysis KatG numbering). The known crystal structures suggest that Tyr(249) and Met(275) could be within hydrogen-bonding distance to Arg(439). To investigate the role of this peculiar adduct, the variants Y249F, M275I, R439A, and R439N were investigated by electronic absorption, steady-state and transient-state kinetic techniques and EPR spectroscopy combined with deuterium labeling. Exchange of these conserved residues exhibited dramatic consequences on the bifunctional activity of this peroxidase. The turnover numbers of catalase activity of M275I, Y249F, R439A, and R439N are 0.6, 0.17, 4.9, and 3.14% of wild-type activity, respectively. By contrast, the peroxidase activity was unaffected or even enhanced, in particular for the M275I variant. As shown by mass spectrometry and EPR spectra, the KatG typical adduct is intact in both Arg(439) variants, as is the case of the wild-type enzyme, whereas in the M275I variant the covalent link exists only between Tyr(249) and Trp(122). In the Y249F variant, the link is absent. EPR studies showed that the radical species formed upon reaction of the Y249F and R439A/N variants with peroxoacetic acid are the oxoferryl-porphyrin radical, the tryptophanyl and the tyrosyl radicals, as in the wild-type enzyme. The dramatic loss in catalase activity of the Y249F variant allowed the comparison of the radical species formed with hydrogen peroxide and peroxoacetic acid. The EPR data strongly suggest that the sequence of intermediates formed in the absence of a one electron donor substrate, is por(.-)(+) --> Trp(.-) (or Trp(.-)(+)) --> Tyr(.-). The M275I variant did not form the Trp(.-) species because of the dramatic changes on the heme distal side, most probably induced by the repositioning of the remaining Trp(122)-Tyr(249) adduct. The results are discussed with respect to the bifunctional activity of catalase-peroxidases.     

SF2/ASF regulates proteomic diversity by affecting the balance between translation initiation mechanisms

Post‐splicing activities have been described for a subset of shuttling serine/arginine‐rich splicing regulatory proteins, among them SF2/ASF. We showed that growth factors activate a Ras‐PI 3‐kinase‐Akt/PKB signaling pathway that not only modifies alternative splicing of the fibronectin EDA exon, but also alters in vivo translation of reporter mRNAs containing the EDA binding motif for SF2/ASF, providing two co‐regulated levels of isoform‐specific amplification. Translation of most eukaryotic mRNAs is initiated via the scanning mechanism, which implicates recognition of the m7G cap at the mRNA 5′‐terminus by the eIF4F protein complex. Several viral and cellular mRNAs are translated in a cap‐independent manner by the action of cis‐acting mRNA elements named internal ribosome entry sites that direct internal ribosome binding to the mRNA. Here we use bicistronic reporters that generate mRNAs carrying two open reading frames, one translated in a cap‐dependent manner while the other by internal ribosome entry site‐dependent initiation, to show that in vivo over‐expression of SF2/ASF increases the ratio between cap‐dependent and internal ribosome entry site‐dependent translation. Consistently, knocking‐down of SF2/ASF causes the opposite effect. Changes in expression levels of SF2/ASF also affect alternative translation of an endogenous mRNA, that one coding for fibroblast growth factor‐2. These results strongly suggest a role for SF2/ASF as a regulator of alternative translation, meaning the generation of different proteins by the balance among these two translation initiation mechanisms, and expand the known potential of SF2/ASF to regulate proteomic diversity to the translation field. J. Cell. Biochem. 107: 826–833, 2009. © 2009 Wiley‐Liss, Inc.     

Benthic communities along a littoral of the Central Adriatic Sea (Italy)

Bacteria, meio- and macrofauna were investigated at different depths in a coastal area of the Central Adriatic Sea, yielding information about the composition and abundance of the benthic community. In particular, 14 nematode genera were recorded for the first time in the Upper Adriatic Sea. All communities resulted as being significantly different between inshore and offshore stations, especially when the season interaction was considered. Sediment grain size seemed to be the main natural variable, along with trophic resources, affecting the distribution and composition of these benthic components, whilst there was no clear evidence of competition for food sources and predatory pressure between the communities. Meiofauna appeared the most useful community for detecting disturbances and river influences. In particular, the lowest copepod abundance in the shallow waters seemed to be related to a greater anthropogenic disturbance inshore, whilst meiofaunal abundance and diversity together with the nematode maturity index suggest the influence of the Foglia and Metauro rivers and the small stream Arzilla.     

Linking Environmental Forcing and Trophic Supply to Benthic Communities in the Vercelli Seamount Area (Tyrrhenian Sea)

Seamounts and their influence on the surrounding environment are currently being extensively debated but, surprisingly, scant information is available for the Mediterranean area. Furthermore, although the deep Tyrrhenian Sea is characterised by a complex bottom morphology and peculiar hydrodynamic features, which would suggest a variable influence on the benthic domain, few studies have been carried out there, especially for soft-bottom macrofaunal assemblages. In order to fill this gap, the structure of the meio-and macrofaunal assemblages of the Vercelli Seamount and the surrounding deep area (northern Tyrrhenian Sea – western Mediterranean) were studied in relation to environmental features. Sediment was collected with a box-corer from the seamount summit and flanks and at two far-field sites in spring 2009, in order to analyse the metazoan communities, the sediment texture and the sedimentary organic matter. At the summit station, the heterogeneity of the habitat, the shallowness of the site and the higher trophic supply (water column phytopigments and macroalgal detritus, for instance) supported a very rich macrofaunal community, with high abundance, biomass and diversity. In fact, its trophic features resembled those observed in coastal environments next to seagrass meadows. At the flank and far-field stations, sediment heterogeneity and depth especially influenced the meiofaunal distribution. From a trophic point of view, the low content of the valuable sedimentary proteins that was found confirmed the general oligotrophy of the Tyrrhenian Sea, and exerted a limiting influence on the abundance and biomass of the assemblages. In this scenario, the rather refractory sedimentary carbohydrates became a food source for metazoans, which increased their abundance and biomass at the stations where the hydrolytic-enzyme-mediated turnover of carbohydrates was faster, highlighting high lability.     

Benthic communities on a sandy Ligurian beach (NW Mediterranean)

The different components of the benthic community of a sandy microtidal beach (Arenzano) in Liguria (NW Mediterranean) were investigated during late spring (May) 2002 and 2003. Sampling was carried out in two transects, chosen in order to represent the characteristics of the entire beach and their eventual spatial variations. Each transect included two stations: one placed in the swash zone (SW) and one in the surf zone (SF). Although no significant differences were found in the sediment texture over the 2 years (t-tests p > 0.1 for all the granulometric fractions), notwithstanding an increase in the mean grain size (from 0.8 to 1.1 mm) between the sampling periods, 2002 was characterised by a higher quantity of organic matter (on average 14.4 vs. 3.6 gC m⁻² for the sum of proteins, carbohydrates and lipids) and higher bacterial biomass (on average 1.9 vs. 0.9 gC m⁻²). The metazoan assemblages (meiofauna and macrofauna) were also richer (density = 2.9 × 10⁵ vs. 1.0 × 10⁵ ind. m⁻², biomass = 0.09 vs. 0.03 gC m⁻² for meiofauna; density = 1988 vs. 739 ind. m⁻², biomass = 0.14 vs. 0.03 gC m⁻² for macrofauna) in 2002. A significant quantitative reduction (t-test for proteins, carbohydrates and lipids, at least p = 0.004) in the food supply in 2003 affected the abundance of the metazoans, as confirmed by a multivariate analysis that clearly differentiated the 2 years, and seemed to inhibit their relationships within the benthic food web. The bacterial biomass was always dominant, even under the least favourable trophic conditions, due to the ability of bacteria to adapt to a very harsh environment. Our results suggest that the food supply played an important role in the benthic community structures of the beach during late spring, bacteria being the key organisms within the benthic system. The communities seemed to be bottom-up controlled, while predation seemed to be irrelevant.     

Roles for Arg426 and Trp111 in the modulation of NADH oxidase activity of the catalase-peroxidase KatG from Burkholderia pseudomallei inferred from pH-induced structural changes

Crystals of Burkholderia pseudomallei KatG retain their ability to diffract X-rays at high resolution after adjustment of the pH from 5.6 to 4.5, 6.5, 7.5, and 8.5, providing a unique view of the effect of pH on protein structure. One significant pH-sensitive change lies in the appearance of a perhydroxy group attached to the indole nitrogen of the active site Trp111 above pH 7, similar to a modification originally observed in the Ser324Thr variant of the enzyme at pH 5.6. The modification forms rapidly from molecular oxygen in the buffer with 100% occupancy after one minute of soaking of the crystal at room temperature and pH 8.5. The low temperature (4 K) ferric EPR spectra of the resting enzyme, being very sensitive to changes in the heme iron microenvironment, confirm the presence of the modification above pH 7 in native enzyme and variants lacking Arg426 or Met264 and its absence in variants lacking Trp111 or Tyr238. The indole-perhydroxy group is very likely the reactive intermediate of molecular oxygen in the NADH oxidase reaction, and Arg426 is required for its reduction. The second significant pH-sensitive change involves the buried side chain of Arg426 that changes from one predominant conformation at low pH to a second at high pH. The pH profiles of the peroxidase, catalase, and NADH oxidase reactions can be correlated with the distribution of Arg426 conformations. Other pH-induced structural changes include a number of surface-situated side chains, but there is only one change involving a displacement of main chain atoms triggered by the protonation of His53 in a deep pocket in the vicinity of the molecular 2-fold axis.