Efficient Screening of Marine Extracts for Protease Inhibitors by Combining FRET Based Activity Assays and Surface Plasmon Resonance Spectroscopy Based Binding Assays

The screening of extracts from marine organisms is a widely used strategy to discover new drug leads. A common problem in the screening process is the generation of false positive hits through unspecific effects from the complex chemical composition of the crude extracts. In this study, we explored a combination of a fluorescence resonance energy transfer (FRET) based activity assay and a surface plasmon resonance (SPR) based binding assay to avoid this problem. An aqueous extract was prepared from rest raw material of the Norwegian spring spawning herring, and further fractionated by methanol solubility and solid phase extraction. FRET based activity assays were used to determine the influence of each extract on the activity of different proteases. Several extracts showed more than 50% inhibition. The inhibition mechanisms were elucidated by SPR based competition experiments with known inhibitors. For the secreted aspartic proteases 1, 2, 3 and HIV-1 protease, the results indicated that some extracts contain inhibitors interacting specifically with the active site of the enzymes. The study shows that a combination of an activity assay and an SPR based binding assay is a powerful tool to identify potent inhibitors in marine extracts. Furthermore, the study shows that marine vertebrates offer an interesting source for new bioactive compounds, although they have rarely been explored for this purpose.     

Immunocytochemical localization of the Bacillus sphaericus binary toxin components in Culex quinquefasciatus (Diptera: Culicidae) larvae midgut

This study describes the immunocytochemical localization of the Bacillus sphaericus 2362 binary toxin components, BinA and BinB, in Culex quinquefasciatus larvae that had been intoxicated with this entomopathogen. Ultrathin sections of C. quinquefasciatus larvae midgut embedded in the hydrophilic resin L.R. White were incubated with the antibodies anti-BinA or anti-BinB and then revealed with goat anti-rabbit IgG coupled to gold particles. Immunocytochemical detection demonstrated the presence of specific labeling in ultra-thin sections that had been incubated with the BinA antiserum. Gold particles were detected on the apical areas of cell membranes and inside the epithelial cell cytoplasm, particularly the mitochondria, of cells from the gastric caeca and posterior stomach in larvae exposed during 2 or 24 h to the entomopathogen. A similar labeling pattern was observed in ultrathin sections from both midgut regions when incubated with BinB antiserum.     

CD46 in meningococcal disease

The human-specific bacterial pathogen Neisseria meningitidis is a major cause of sepsis and/or meningitis. The pili of N. meningitidis interact with CD46, a human cell-surface protein involved in regulation of complement activation. Transgenic mice expressing human CD46 were susceptible to meningococcal disease, because bacteria crossed the blood-brain barrier in these mice. Development of disease was more efficient with piliated bacteria after intranasal, but not intraperitoneal, challenge of CD46 transgenic mice, suggesting that human CD46 facilitates pilus-dependent interactions at the epithelial mucosa. Hence, the human CD46 transgenic mice model is a potentially useful tool for studying pathogenesis and for vaccine development against meningococcal disease.     

Comparative study of the characteristics of the carcass and the meat of the Chato Murciano pig and its cross with Iberian pig, reared indoors

The Chato Murciano pig is a breed catalogued as a Special Protection breed in danger of extinction. At present it is commonly reared in an intensive system. We have studied the mineral composition and other characteristics of its carcass and meat. Samples from the Musculus longissimus lumborum were taken from a total number of 43 animals, divided into two groups, Chato Murciano and Chato Murciano crossed with Iberian, reared in a conventional indoor system. The resulting meat of the Chato Murciano pig and its cross with Iberian was especially rich in iron and copper, rich in phosphorus, and marginally poor in calcium and sodium. The evolution of the pH of the carcass was that of pigs which do not produce PSE (pale, soft and exudative) meat. Some relevant positive correlation coefficients between minerals (P ≤ 0.05) have been found.     

Cadmium Effects in Sunflower: Nutritional Imbalances in Plants and Calluses

The effects of cadmium (Cd) exposure on sunflower (Helianthus annuus L.) nutrient accumulation remain unclear. However, studies concerning crop improvement for Cd tolerance suggest the use of biotechnology techniques such as tissue culture. It is still unknown whether in vitro cells respond to Cd exposure in a way similar to plants. In this paper, the objectives were (1) to characterize the effects of Cd exposure in macronutrient and micronutrient accumulation in different sunflower organs/tissues and (2) to compare the behavior of two culture systems (plants vs. tissue culture) regarding Cd and nutrient accumulation. To achieve these aims, sunflower plants were grown hydroponically in the presence of Cd (at levels of 0, 5, 50, and 500 μ M). For in vitro cultures, seeds were germinated axenically and leaf explants were then grown on Murashige and Skoog medium (MS). One-month-old calluses were grown on MS medium containing 0, 5, 50, and 500 μ M Cd. After 21 d of exposure to 500 μ M, all plants were dead. The contents of macro- and micronutrients and of Cd were determined by ICPS in 18 d-exposed plants and calluses and in calluses exposed for six months to 50 μ M Cd. At day 18, Cd content increased in leaves, roots, and calluses. Cadmium exposure also decreased the contents of magnesium (Mg), calcium (Ca), iron (Fe), and manganese (Mn) in roots and of Mg, Ca, copper (Cu), Fe, and Mn in shoots. Exposed calluses suffered decreases only in Mg, Ca, and Mn contents. The contents of most of these nutrients in six-month-exposed calluses were similar to those of the control calluses, indicating that these long-term exposed in vitro cells developed mechanisms for regulating the effects of Cd on the accumulation of nutrients.     

Habitat characterization,anthropogenic impacts and conservation of rhodolith beds off southeastern Malta

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Effect of rice growth stages and silicon on sheath blight development

ABSTRACT The objective of this study was to determine the effect of silicon (Si) and rice growth stages on tissue susceptibility to sheath blight (Rhizoctonia solani Kühn) under controlled conditions. Rice plants (cv. Rio Formoso) were grown in pots containing low-Si soil amended with Si at 0, 0.48, 0.96, 1.44, and 1.92 g pot(-1) and inoculated with R. solani at the following days after emergence: 45 (four-leaf stage), 65 (eight-leaf stage), 85 (tillering), 117 (booting), and 130 (panicle exsertion). For plants inoculated with R. solani at all growth stages, Si concentration in straw increased as rate of Si increased from 0 to 1.92 g pot(-1). Concentration of calcium in the straw did not differ among plant growth stages. Although incubation period was not affected by the amount of Si added to the soil, this variable was shorter at booting and panicle exsertion stages. As the rates of Si increased in the soil, the total number of sheath blight lesions on sheaths and total area under the relative lesion extension curve decreased at all plant growth stages. The severity of sheath blight was lower at booting and panicle exsertion stages as the rates of Si increased in the soil. In general, plants grown in Si-nonamended pots and inoculated with R. solani were more vulnerable to infection at all growth stages, but especially at 45 days after emergence. Plant dry weights for inoculated plants increased as the Si rates increased from 0 to 1.92 g pot(-1). The greatest dry weight increases occurred for plants inoculated at booting and panicle exsertion stages. Si fertilization is a promising method for controlling sheath blight in areas where soil is Si deficient and when cultivars that exhibit an acceptable level of resistance to sheath blight are not available for commercial use.