Parasites, condition, immune responsiveness and carotenoid-based ornamentation in male red-legged partridge Alectoris rufa

Sexual ornaments might indicate better condition, fewer parasites or a greater immune responsiveness. Carotenoid-based ornaments are common sexual signals of birds and often influence mate choice. Skin or beaks pigmented by carotenoids can change colour rapidly, and could be particularly useful as honest indicators of an individual's current condition and/or health. This is because carotenoids must be acquired through diet and/or allocation for ornamental coloration might be to the detriment of self-maintenance needs. Here, we investigated whether the carotenoid-based coloration of eye rings and beak of male red-legged partridges Alectoris rufa predicted condition (mass corrected for size), parasite load (more specifically infection by coccidia, a main avian intestinal parasite) or a greater immune responsiveness (swelling response to a plant lectin, phytohaemagluttinin, or PHA). Redness of beak and eye rings positively correlated with plasma carotenoid levels. Also, males in better condition had fewer coccidia, more circulating carotenoids and a greater swelling response to PHA. Carotenoid-based ornamentation predicted coccidia abundance and immune responsiveness (redder males had fewer coccidia and greater swelling response to PHA), but was only weakly positively related to condition. Thus, the carotenoid pigmentation of beak and eye rings reflected the current health status of individuals. Our results are consistent with the hypothesis that allocation trade-offs (carotenoid use for ornamentation versus parasite defence needs) might ensure reliable carotenoid-based signalling.     

Morphological and anatomical evaluation of adult and juvenile leaves of olive plants

The olive tree (Olea europaea L.), like many other woody plants, has a long juvenile period in which the plant is not able to produce flowers. Knowledge of the moment when the plant is capable of flowering is important for breeding programs and also for determining the physiological basis for sexual reproductive behavior, but currently the only indicator of that moment is the actual flowering. In many species, the juvenile-to-adult phase shift includes changes in leaf structure known as heteroblasty, that is, varied form of successive leaves on the same plant. Some differences have been observed between juvenile and adult olive leaves, particularly in size and form, but to our knowledge, no complete systematic study has been carried out. In this research, we measured size, morphology and anatomy for juvenile and adult leaves of olive plants grown from seeds. Differences were found in most of the parameters studied, including leaf size, form, mesophyll thickness, layers of palisade parenchyma and quantity of peltate trichomes, which were generally significant but overlapping between the two leaf types. The most consistent and striking difference was the presence of an organized layer of subepidermal cells only in the abaxial mesophyll of adult leaves. This characteristic could be a simple and effective criterion of phase change in the olive tree.     

A multiple approach for the evaluation of the spatial distribution and dynamics of a forest habitat: the case of Apennine beech forests with Taxus baccata and Ilex aquifolium

An approach integrating phytosociological and stand structure surveys with the predictive modelling of species distribution was applied to analyse the spatial distribution and dynamics of the Apennine beech forests with Taxus and Ilex, a high conservation priority forest habitat in Europe. The homogeneity of the habitat was tested trough a Mann–Whitney test between beech woods with Taxus and those with Ilex with respect to climatic, topographic, structural and environmental parameters: the former have proven to be more microthermic, mesophilous and characterised by a closer canopy. Five statistical models were compared to analyse the relationship between bioclimatic parameters and Taxus and Ilex spatial distribution: Regression Tree Analysis, the most efficient model, has shown that the distribution of Taxus is influenced by precipitation variables, while Ilex is mainly influenced by temperature variables. This model highlighted that Ilex has a potential area that surrounds, at lower altitudes, that of Taxus. A stepwise multiple regression analysis has been applied to identify the factors influencing the regeneration of the two species: beside climatic parameters, Taxus regeneration is negatively influenced by soil nitrate concentration (an indicator of livestock disturbance) while Ilex is negatively influenced by beech forest cover. Traditional management practices seem to have an effect on the regeneration of the two species: frequent cuts favour the regeneration of Ilex, reducing the forest cover and allowing more light penetration, while Taxus, less resistant to grazing livestock, is confined to more inaccessible places. The multiple approach has proven to be useful for the elaboration of two differentiated conservation strategies for the two beech forest types.     

Antimutagenic and antioxidant activities of quebracho phenolics (Schinopsis balansae) recovered from tannery wastewaters

Quebracho extracts are used in tannery due to their high concentration of phenolics. The Mexican tannery industry uses around 450 kg/m(3) of which, 150 kg/m(3) remains in wastewaters and are discharged in drain pipe systems or rivers. The quebracho phenolics recovered from tannery wastewater (QPTW) was characterized by HPLC. The antimutagenic and antioxidant activities as well as the microbiological quality were evaluated. Total phenolic content of QPTW was 621mg catechin equivalent/g sample. Gallic and protocatechuic acids were the major components characterized by HPLC. QPTW showed an inhibition range on aflatoxin B(1) mutagenicity from 16 to 60% and was dose-dependent. Antioxidant activity (defined as beta-carotene bleaching) of QPTW (64.4%) at a dose of 12.3mg/mL was similar to that of BHT (68.7%) at a dose of 0.33 mg/mL, but lower than Trolox (90.8% at a dose of 2.5mg/mL); meanwhile antiradical activity (measured as reduction of DPPH) (60.8%) was higher than that of BHT (50.8%) and Trolox (34.2%). Quebracho residues were demonstrated to be an outstanding source of phenolic acids and for research and industrial uses.     

The proteoglycan region of the tumor-associated carbonic anhydrase isoform IX acts as anintrinsic buffer optimizing CO2 hydration at acidic pH values characteristic of solid tumors

The enzymatic activities of carbonic anhydrase (CA, EC 4.2.1.1) isozymes CA I, II, IX (catalytic domain (cdCA IX) and catalytic domain plus proteoglycan, flCA IX), XII and XIV were investigated as a function of pH for the CO₂ hydration to bicarbonate and a proton. The cytosolic isoforms CA I and II as well as the catalytic domain of CA IX, together with the transmembrane isoforms CA XII and XIV showed sigmoid pH dependencies of k_cat/K_M, with a pK_a of 6.90–7.10, showing thus optimal catalytic efficiency around pH 7. The full length CA IX had a similar shape of the pH dependency curve but with a pK_a of 6.49, having thus maximal catalytic activity at pH values around 6.5, typical of hypoxic solid tumors in which CA IX is overexpressed. The proteoglycan domain of CA IX (present only in this transmembrane isoform) may thus act as an intrinsic buffer promoting efficient CO₂ hydration at acidic pH values found in hypoxic tumors.     

Carbonic anhydrase inhibitors: The membrane-associated isoform XV is highly inhibited by inorganic anions

The membrane-associated mouse isozyme of carbonic anhydrase XV (mCA XV), has been investigated for its interaction with anion inhibitors. mCA XV is an isoforms possessing a very particular inhibition profile by anions, dissimilar to that of all other mammalian CAs investigated earlier. Many simple inorganic anions (thiocyanate, cyanide, azide, bicarbonate, hydrogen sulfide, bisulfite and sulfate) showed low micromolar inhibition constants against mCA XV (K_Is of 8.2–10.1 μM), whereas they acted as much weaker (usually millimolar) inhibitors of other isoforms. Halides, nitrate, nitrite, carbonate, sulfamate, sulfamide and phenylboronic/arsonic acid were weaker inhibitors, with inhibition constants in the range of 27.6–288 μM. Our data may be useful for the design of more potent inhibitors of mCA XV (considering various zinc binding groups present in the anions investigated here, e.g., the sulfonate one) and for understanding some physiologic/pharmacologic consequences of mCA XV inhibition by anions such as bicarbonate or sulfate which show quite high affinity for it.     

Carbonic anhydrase inhibitors. The nematode α-carbonic anhydrase of Caenorhabditis elegans CAH-4b is highly inhibited by 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulfonamides

A series of 2-(hydrazinocarbonyl)-3-substituted-phenyl-1H-indole-5-sulfonamides possessing various 2-, 3- or 4-substituted phenyl groups with methyl-, halogeno- and methoxy-functionalities, as well as the perfluorophenyl moiety, have been evaluated as inhibitors of an α-carbonic anhydrase (CA, EC 4.2.1.1) of the nematode model organism Caenorhabditis elegans (CAH-4b, or ceCA). The substitution pattern at the 3-phenyl ring highly influenced the ceCA inhibitory activity of these heterocyclic sulfonamides, with best inhibitors (K_Is in the range of 6.0–13.4 nM) incorporating 3-methyl-, 4-methyl-, 2-/3-/4-fluoro-, 4-chloro- and 3-/4-bromo-phenyl such moieties. Some of these sulfonamides also showed a good selectivity profile for the inhibition of the nematode over the human isozymes CA I and II (selectivity ratios in the range of 1.78–4.95 for the inhibition of ceCA over hCA II). These data can be used for the design of possibly new antihelmintic drugs, since the genome of many parasitic nematodes encode for a multitude of orthologue CA isozymes to ceCA investigated here.     

On the role of Posidonia oceanica beach wrack for macroinvertebrates of a Tyrrhenian sandy shore

The use of Posidonia oceanica beach wrack by macroinvertebrates of the sandy beach at Burano (Tuscany, Italy) was assessed by following the colonisation dynamics of the wrack and analysing the stable isotopes ‘scenario’ of the main local carbon and nitrogen sources and consumers. One-hundred experimental cylinders, filled with P. oceanica wrack, were placed on the beach and sampled over a 1-month period. Abundance and species richness of macroinvertebrates in wracks varied through time. Wrack was colonised by crustaceans almost immediately after deployment of the experimental cylinders. The amphipod Talitrus saltator largely dominated the faunal assembly and, together with the isopod Tylos europaeus, occupied the wracks closer to the sealine. These were followed by dipterans, staphylinids, pselaphids and tenebrionids that occurred in drier wracks higher up on the eulittoral. Moisture content of the wrack and sand decreased through space and time. This was the primary factor explaining the spatial and temporal changes observed in macroinvertebrate abundance, with species colonising or abandoning wracks according to thresholds of environmental parameters. Isotopic analysis clearly established the absence of any direct dietary link between P. oceanica wrack and macroinvertebrates. Terrestrial food sources were also discarded. Both our experimental data and a literature search showed that the organic matter from seston as filtered by the sand is the most plausible carbon and nitrogen source for beach food webs. Even if P. oceanica wrack is not a trophic source for macroinvertebrates, it is vitally important as a physical structure that provides detritivorous and predatory species with refuge from environmentally stressful conditions.     

Regenerative pharmacology in the treatment of genetic diseases: the paradigm of muscular dystrophy

Current evidence supports the therapeutic potential of pharmacological interventions that counter the progression of genetic disorders by promoting regeneration of the affected organs or tissues. The rationale behind this concept lies on the evidence that targeting key events downstream of the genetic defect can compensate, at least partially, the pathological consequence of the related disease. In this regard, the beneficial effect exerted on animal models of muscular dystrophy by pharmacological strategies that enhance muscle regeneration provides an interesting paradigm. In this review, we describe and discuss the potential targets of pharmacological strategies that promote regeneration of dystrophic muscles and alleviate the consequence of the primary genetic defect. Regenerative pharmacology provides an immediate and suitable therapeutic opportunity to slow down the decline of muscles in the present generation of dystrophic patients, with the perspective to hold them in conditions such that they could benefit of future, more definitive, therapies.     

Tetramerization and Cooperativity in Plasmodium falciparum Glutathione S-Transferase Are Mediated by Atypic Loop 113�C119

Glutathione S-transferase of Plasmodium falciparum (PfGST) displays a peculiar dimer to tetramer transition that causes full enzyme inactivation and loss of its ability to sequester parasitotoxic hemin. Furthermore, binding of hemin is modulated by a cooperative mechanism. Site-directed mutagenesis, steady-state kinetic experiments, and fluorescence anisotropy have been used to verify the possible involvement of loop 113–119 in the tetramerization process and in the cooperative phenomenon. This protein segment is one of the most prominent structural differences between PfGST and other GST isoenzymes. Our results demonstrate that truncation, increased rigidity, or even a simple point mutation of this loop causes a dramatic change in the tetramerization kinetics that becomes at least 100 times slower than in the native enzyme. All of the mutants tested have lost the positive cooperativity for hemin binding, suggesting that the integrity of this peculiar loop is essential for intersubunit communication. Interestingly, the tetramerization process of the native enzyme that occurs rapidly when GSH is removed is prevented not only by GSH but even by oxidized glutathione. This result suggests that protection by PfGST against hemin is independent of the redox status of the parasite cell. Because of the importance of this unique segment in the function/structure of PfGST, it could be a new target for the development of antimalarial drugs.Approximately two million deaths in the world per year are caused by Plasmodium falciparum, the parasite responsible for tropical malaria (1, 2). In the last years, increasing interest has been developing for the peculiar glutathione S-transferase (PfGST)³ expressed by this parasite. Expressed in almost all living organisms, GSTs represent a large superfamily of multifunctional detoxifying enzymes that are able to conjugate GSH to a lot of toxic electrophilic compounds, thus facilitating their excretion. Many other protection roles of GSTs have been described, including the enzymatic reduction of organic peroxides (3–5), the inactivation of the proapoptotic JNK through a GST·JNK complex (6), and the protection of the cell from excess nitric oxide (7). The mammalian cytosolic GSTs are dimeric proteins grouped into eight species-independent classes termed Alpha, Kappa, Mu, Omega, Pi, Sigma, Theta, and Zeta on the basis of sequence similarity, immunological reactivity, and substrate specificity (3, 8–11). PfGST is one of the most abundant proteins expressed by P. falciparum (from 1 to 10%, i.e. from 0.1 to 1 mm) (12), and different from what occurs in many organisms, it is the sole GST isoenzyme expressed by this parasite. Despite its structural similarity to the Mu class GST, this specific isoenzyme cannot be assigned to any known GST class (13). The interest in this enzyme is due to its particular protective role in the parasite. In fact, in addition to the usual GST activity that promotes the conjugation of GSH to electrophilic centers of toxic compounds, this protein efficiently binds hemin, and thus it could protect the parasite (that resides in the erythrocytes) from the parasitotoxic effect of this heme by-product (14). Specific compounds that selectively inhibit its catalytic activity or hemin binding could be promising candidates as antimalarial drugs. In this context, the discovery of structural or mechanistic properties of this enzyme that are not found in other GSTs may be important for designing selective inhibitors that are toxic to the parasite but harmless for the host cells. Two properties never observed in other members of the GST superfamily are of particular interest. The first property is that this enzyme, in the absence of GSH, is inactivated in a short time and loses its ability to bind hemin (15). Recent studies indicated that the inactivation process is related to a dimer to tetramer transition (13, 16, 17). The second property is the strong positive homotropic phenomenon that modulates the affinity of the two subunits for hemin (15). The x-ray crystal structure of PfGST, solved by two different groups (13, 18), provides insights into this effect. From a structural point of view, the most intriguing differences of PfGST when compared with other GSTs are a more solvent-exposed H-site and an atypic extra loop connecting helix α-4 and helix α-5 (residues 113–119; see also Fig. 1) that could be involved in the dimer-dimer interaction. Actually, in the absence of ligands, two biological dimers form a tetramer, and these homodimers are interlocked with each other by loop 113–119 of one homodimer, which occupies an H-site of the other homodimer (13, 18). Upon binding of S-hexylglutathione, loop 113–119 rearranges; residues Asn-114, Leu-115, and Phe-116 form an additional coil in helix α-4; and the side chains of Asn-111, Phe-116, and Tyr-211 flip into the H-site of the same dimer (17, 18). The changed course of residues 113–119 in the liganded enzyme prevents the interlocking of the dimers.Open in a separate windowFIGURE 1.A, structural changes of loop 113–119 occurring in the dimer (light blue model and yellow loop; Protein Data Bank code 2AAW) to tetramer (blue model and orange loop; Protein Data Bank code 1OKT) transition. Red spheres indicate the amino acids replaced in this study to obtain mutants A, B, and C. B, model of hemin·PfGST complex obtained by docking simulation using the crystal structure for Protein Data Bank code 1Q4J (15). Hemin is shown in red, loop 113–119 is in orange, and GSH is shown as yellow sticks.In this paper, by means of site-directed mutagenesis, fluorescence anisotropy, kinetic studies, and size exclusion chromatography, we check the influence of selected mutations of this atypic loop in the tetramerization process and the possible involvement of this protein segment in the cooperative phenomenon that characterizes hemin binding. In addition we describe that the tetramerization process is inhibited not only by GSH but even by GSSG. This finding suggests that hemin binding of PfGST is independent of the redox status of the cell. Finally, we demonstrate that the presence of GSH (or GSSG) in the active site is not essential for hemin binding, but this interaction only requires an active dimeric conformation.