A chitin-like component in Aedes aegypti eggshells, eggs and ovaries

An insoluble white substance was prepared from extracts of eggshells of Aedes aegypti, the yellow fever mosquito and dengue vector. Its infrared and proton NMR spectra were similar to that of standard commercial chitin. This putative chitin-like material, also obtained from ovaries, newly laid and dark eggs, was hydrolyzed in acid and a major product was identified by HPLC to be glucosamine. The eggshell acid hydrolysate was also analyzed by ESI-MS and an ion identical to a glucosamine monoprotonated species was detected. The presence of chitin was also analyzed during different developmental stages of the ovary using a fluorescent microscopy technique and probes specific for chitin. The results showed that a chitin-like material accumulates in oocytes during oogenesis. Streptomyces griseus chitinase pre-treatment of oocytes greatly reduced the chitin-derived fluorescence. Chitinase activity was detected in newborn larvae and eggs prior to hatching. Feeding experiments indicated that the chitin synthesis inhibitor lufenuron inhibited chitin synthesis, either when mosquitoes were allowed to feed directly on lufenuron-treated chickens or when an artificial feeding system was used. Lufenuron inhibited egg hatch, larval development and reduced mosquito viability. These data demonstrate for the first time that (1) a chitin-like material is present in A. aegypti eggs, ovaries and eggshells; (2) a chitin synthesis inhibitor can be used to inhibit mosquito oogenesis; and (3) chitin synthesis inhibitors have potential for controlling mosquito populations.     

Radioiodination of new EGFR inhibitors as potential SPECT agents for molecular imaging of breast cancer

In our search for the development of novel SPECT radioligands for EGFR positive tumours, new potentially irreversible tyrosine kinase (TK) inhibitors are being explored. The radioiodination of N-{4-[(3-chloro-4-fluorophenyl) amino]quinazoline-6-yl}-3-bromopropionamide, a novel EGFR-TK inhibitor synthesised in our laboratory, was accomplished via halogen exchange. Purification by RP-HPLC gave [125I]-N-{4-[(3-chloro-4-fluorophenyl)amino]quinazoline-6-yl}-3-iodopropionamide with a radiochemical purity higher than 95% and a high specific activity. In vitro studies indicate that both iodinated quinazoline and its bromo precursor inhibit A431 cell growth and also possess higher potency than the parent quinazoline to inhibit the EGFR autophosphorylation. In vivo stability studies suggest metabolization of the radioiodinated quinazoline indicating a short biological half-life. The in vitro results point out that these quinazoline derivatives could be promising candidates for SPECT imaging of EGFR positive tumours provided that they are selectively modified in order to achieve better in vivo radiochemical stability.     

Inhibition of S. aureus alpha-hemolysin and B. anthracis lethal toxin by beta-cyclodextrin derivatives

Many pathogens utilize the formation of transmembrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered to be important virulence factors, which makes them attractive targets for the discovery of new therapeutic agents. Our research is based on the idea that compounds designed to block the pores can inhibit the action of virulence factors, and that the chances to find high affinity blocking agents increase if they have the same symmetry as the target pore. Recently, we demonstrated that derivatives of beta-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. To test the broader applicability of this approach, we sought beta-cyclodextrin derivatives capable of inhibiting the activity of Staphylococcus aureus alpha-hemolysin (alpha-HL), which is regarded as a major virulence factor playing an important role in staphylococcal infection. We identified several amino acid derivatives of beta-cyclodextrin that inhibited the activity of alpha-HL and LeTx in cell-based assays at low micromolar concentrations. One of the compounds was tested for the ability to block ion conductance through the pores formed by alpha-HL and PA in artificial lipid membranes. We anticipate that this approach can serve as the basis for a structure-directed drug discovery program to find new and effective therapeutics against various pathogens that utilize pore-forming proteins as virulence factors.     

Environmental Correlates for Species Richness among Amphibians and Reptiles in a Climate Transition Area

Correlations between environmental factors and the distribution of amphibian and reptile species richness were investigated in a climate transition area, Peneda-Gerês National Park (PNPG), in North-Western Portugal. Using presence-data at a local-scale (1 × 1 km), Ecological-Niche Factor Analysis (ENFA) identified a mixture of climatic (precipitation and number of days with fog), topographical (altitude and relief) and habitat factors (number of watercourses and water surfaces, the type of the largest water surface and tree diversity cover), as accurate predictors of species occurrence. Three factors were common for both taxonomic groups, and consistently presented a positive relation with species occurrence: precipitation, number of water surfaces, and tree diversity cover; suggesting a strong coincidence in the environmental correlates that influence amphibian and reptile species richness. Distribution patterns of observed and predicted species richness were compared using a Geographical Information System. Overall, three high species richness areas were predicted in common for both taxonomic groups and two additional areas for amphibians only. These areas matched with the observed species richness but suggested larger areas of high species richness. The location of the PNPG in a biogeographic crossroad, between Euro-Siberian and Mediterranean provinces, emphasised species richness of amphibians and reptiles and suggests a high priority conservation status for this protected area. Most of Central-Northern Portugal is located in a climatic transition area; therefore, increased species richness should be expected for other areas. Local scale studies for other protected areas should be planned as a framework for the development of multi-scale conservation planning by Portuguese authorities.     

PDE-10A inhibitors as insulin secretagogues

Modulation of cAMP levels has been linked to insulin secretion in preclinical animal models and in humans. The high expression of PDE-10A in pancreatic islets suggested that inhibition of this enzyme may provide the necessary modulation to elicit increased insulin secretion. Using an HTS approach, we have identified quinoline-based PDE-10A inhibitors as insulin secretagogues in vitro. Optimized compounds were evaluated in vivo where improvements in glucose tolerance and increases in insulin secretion were measured.     

Cross-presentation of caspase-cleaved apoptotic self antigens in HIV infection

We found that the proteome of apoptotic T cells includes prominent fragments of cellular proteins generated by caspases and that a high proportion of distinct T cell epitopes in these fragments is recognized by CD8+ T cells during HIV infection. The frequencies of effector CD8+ T cells that are specific for apoptosis-dependent epitopes correlate with the frequency of circulating apoptotic CD4+ T cells in HIV-1-infected individuals. We propose that these self-reactive effector CD8+ T cells may contribute to the systemic immune activation during chronic HIV infection. The caspase-dependent cleavage of proteins associated with apoptotic cells has a key role in the induction of self-reactive CD8+ T cell responses, as the caspase-cleaved fragments are efficiently targeted to the processing machinery and are cross-presented by dendritic cells. These findings demonstrate a previously undescribed role for caspases in immunopathology.     

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.     

Influence of diets supplemented with vitamins C and E on pirarucu (Arapaima gigas) blood parameters

This study evaluated the influence of diets supplemented with 500, 800, 1200 mg kg^− 1 of vitamin C (ascorbic acid or AA) and vitamin E (α-tocopherol or α-T) on the physiological responses of pirarucu fed for 2 months. Weight and mortality were not affected by dietary vitamin type or their concentrations. Significant increase (p < 0.05) on the red blood cells count was obtained on treatments with 800 and 1200 mg AA kg^− 1 and on the hemoglobin concentration on treatment with 500 mg α-T kg^− 1 relatively to control. Mean corpuscular volume presented a significant decrease (p < 0.05) on treatment with 800 and 1200 mg AA kg⁻¹ when compared to control. Mean corpuscular hemoglobin concentration was significantly high (p < 0.05) on treatment with 500 mg α-T kg^− 1. Only in vitamin C treatments, we noticed a significant increase (p < 0.05) in the number of leucocytes relative to control. All fish in the vitamin-supplemented treatments, except 500 mg AA kg^− 1, had high total protein values compared to control. Fish treated with 800 or 1200 mg α-T kg^− 1 also showed increases in plasma glucose concentrations. Our results suggest that 800 and 1200 mg AA kg^− 1 are probably the most suitable concentrations for pirarucu diets, although high vitamin E diets are not necessary for quantitative leucocyte increases for this species.     

False positive galactomannan results in adult hematological patients treated with piperacillin-tazobactam

In this prospective study including 78 adult patients with haematological malignancy (90 episodes) we performed galactomannan (GM) (Platelia Aspergillus) screening twice weekly for the diagnosis of invasive aspergillosis. There were five proven and four probable invasive aspergillosis cases. The sensitivity, specificity and positive and negative predictive values were 100, 88, 47 and 100%, respectively. There were eight patients with false positive GM (10.2%). In six patients the false GM reactivity was due to the administration of piperacillin-tazobactam (P-T). A significant association was found between false positive GM (= or > 0.5) and the administration of P-T (p < 0.01). Two other patients with no invasive aspergillosis (2.5%) and false GM reactivity had graft versus host disease (GVHD) and one of them had also mucositis grade IV. The kinetic patterns of false positive GM due to P-T is discussed.     

Effects of moderate beer consumption on first-line immunity of healthy adults

Moderate alcohol consumption has shown to induce benefits on host specific (cell-mediated and humoral) immune system, but there is scarce literature regarding first-line immune responses. The aim of this study was to investigate differences in non-specific immunity after alcohol abstention and moderate beer consumption in healthy adults. After a 30 day-alcohol abstemious period, 57 healthy volunteers were submitted to a daily moderate consumption of beer (330 mL for women and 660 mL for men, respectively) during the following 30 days. White blood cell counts and phagocytic and oxidative burst activity were evaluated at three points: a) basal, b) abstemious, c) after moderate consumption of beer. Absolute values of leukocytes, neutrophils, lymphocytes and basophiles (x10(9)/L) increased significantly in women from point b to point c (6.34 +/- 1.26 vs. 7.27 +/- 1.97, 3.43 +/- 0.88 vs. 4.13 +/- 1.53, 2.14 +/- 0.50 vs. 2.38 +/- 0.63, and 0.05 +/- 0.02 vs. 0.06 +/- 0.03, respectively; p < 0.05) as well as basophils in men (0.05 +/- 0.03 vs. 0.06 +/- 0.03). A significant increase of oxidative burst capacity was also observed after the moderate consumption of beer in both women (33.90 +/- 19.00 vs. 48.86 +/- 21.83) and men (27.39 +/- 18.13 vs. 39.25 +/- 24.53). In healthy adults, after 30 days of moderate beer consumption the parameter describing the non-specific immunity improved when compared to the basal situation. For several of these parameters, the response is more enhanced in women.