The activity of platelet activating factor-acetyl hydrolase (PAF-AH) in the salivary glands of Rhodnius prolixus

In this work, we investigated the activity of the platelet activating factor acetyl hydrolase (PAF-AH) in the salivary gland homogenates and saliva of Rhodnius prolixus. PAF-AH activity in the salivary gland homogenates was lower than in the saliva. Preliminary characterization of the enzyme demonstrated that it hydrolyzed the substrate 2-thio-PAF, was detectable just in 1 pair of salivary gland homogenates in 0.5 ml buffer, and was stable under different conditions. PMSF, TPCK, TLCK, pepstatin A and p-BPB all inhibited the PAF-AH activity. Enzyme specific activity in salivary gland homogenates diminished immediately after feeding of 5th-instar larvae, and increased before feeding by adult insects. 2-Thio-PAF induced platelet-aggregation that was inhibited by previous incubation of the substrate with salivary gland homogenates or saliva. The relevance of PAF-AH for providing Rhodnius with a feeding mechanism for facilitating the sucking of a high volume of blood meal in a short period is discussed.     

Synthesis and biological characterisation of novel dithiocarbamate containing 5-nitroimidazole Tc-complexes as potential agents for targeting hypoxia

With the aim to develop new potential ^99mTc-radiopharmaceuticals for imaging hypoxia based on the formation of Tc-nitrido complexes, two novel dithiocarbamate containing metronidazole derivatives (L1 and L2) have been prepared and characterised. The synthesis of L1 and L2 was achieved in excellent yield and high purity. Labelling with ^99mTc was successfully performed using a low ligand concentration (approximately 2-3 mg) and the desired products were obtained with high radiochemical purity (>90%). Lipophilicity, plasma protein binding, and biodistribution in normal- and tumour-bearing-CD1 mice studies were performed to asses the potentiality for nuclear medicine oncology. According to the physicochemical and biological behaviour both in healthy animals and in animals bearing solid tumours complex dtcTc1 could be considered as a starting point for the development of new radiopharmaceuticals for imaging hypoxia.     

Throwing the baby out with the bathwater: does laurel forest restoration remove a critical winter food supply for the critically endangered Azores bullfinch?

The invasive Clethra arborea has a dual-role in the diet of the Azores bullfinch, a critically endangered bird species endemic to the island of São Miguel (Azores, Portugal). This is a crucial winter food resource but it lowers the availability of native laurel forest species that compose most of the bird’s diet throughout the year. The removal of this and other invasive alien species is part of current laurel forest habitat restoration programmes, disregarding the impact on the Azores bullfinch population. In order to evaluate the first responses of the Azores bullfinch to habitat restoration, we studied bird diet, foraging behaviour, food availability and habitat occupancy in managed (without C. arborea) and control areas. Significant increases in the availability of native food resources in managed areas were noticeable in the diet, particularly the intake of Ilex perado ssp. azorica and Prunus lusitanica ssp. azorica flower buds. In most of the studied months birds heavily used and foraged in managed over control areas. The one exception was in December, when a resource-gap occurred in managed areas, which may be overcome in the short-term due to re-establishment of native plants following removal of invasive aliens.     

A Thermostable Salmonella Phage Endolysin,Lys68, with Broad Bactericidal Properties against Gram-Negative Pathogens in Presence of Weak Acids

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.     

Effects of Exogenous Recombinant APC in Mouse Models of Ischemia Reperfusion Injury and of Atherosclerosis

Activated protein C (APC) is a serine protease that has both anticoagulant and cytoprotective properties. The cytoprotective effects are protease activated receptor 1 (PAR-1) and endothelial protein C receptor (EPCR) dependent and likely underlie protective effects of APC in animal models of sepsis, myocardial infarction and ischemic stroke. S360A-(A)PC, a variant (A)PC that has no catalytic activity, binds EPCR and shifts pro-inflammatory signaling of the thrombin-PAR-1 complex to anti-inflammatory signaling. In this study we investigated effects of human (h)wt-PC, hS360A-PC, hwt-APC and hS360A-APC in acute (mouse model of acute myocardial ischemia/reperfusion (I/R) injury) and chronic inflammation (apoE^−/− mouse model of atherosclerosis). All h(A)PC variants significantly reduced myocardial infarct area (p<0.05) following I/R injury. IL-6 levels in heart homogenates did not differ significantly between sham, placebo and treatment groups in I/R injury. None of the h(A)PC variants decreased number and size of atherosclerotic plaques in apoE^−/− mice. Only hS360A-APC slightly affected phenotype of plaques. IL-6 levels in plasma were significantly (p<0.001) decreased in hwt-APC and hS360A-PC treated mice. In the last group levels of monocyte chemotactic protein 1 (MCP-1) were significantly increased (p<0.05). In this study we show that both hwt and hS360A-(A)PC protect against acute myocardial I/R injury, which implies that protection from I/R injury is independent of the proteolytic activity of APC. However, in the chronic atherosclerosis model hwt and hS360-(A)PC had only minor effects. When the dose, species and mode of (A)PC administration will be adjusted, we believe that (A)PC will have potential to influence development of chronic inflammation as occurring during atherosclerosis as well.