Enhancement of the inhibitory effect of an IL‐15 antagonist peptide by alanine scanning

IL‐15 is a proinflammatory cytokine that acts early in the inflammatory response and has been associated with several autoimmune diseases including rheumatoid arthritis, where it had been proposed as a therapeutic target. We recently reported an IL‐15 antagonist peptide corresponding to sequence 36–45 of IL‐15 (KVTAMKCFLL) named P8, which specifically binds to IL‐15Rα and inhibits IL‐15 biological activity with a half maximal inhibitory concentration (IC50) of 130 µ m in CTLL‐2 proliferation assay. In order to improve binding of peptide P8 to the receptor IL‐15Rα, we used an Ala scan strategy to study contribution of each individual amino acid to the peptide's antagonist effect. Here, we found that Phe and Cys are important for peptide binding to IL‐15Rα. We also investigated other single site mutations and replaced the second Lys in the sequence by the polar non‐charged amino acid threonine. The resulting peptide [K6T]P8 exhibited a higher activity than P8 with an IC50 of 24 µm . We also found that this peptide was more active than peptide P8 in the inhibition of TNFα secretion by synovial cells from rheumatoid arthritis patients. The peptide [K6T]P8 described in this work is a new type of IL‐15 antagonist and constitutes a potential therapeutic agent for rheumatoid arthritis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Graph Based Study of Allergen Cross-Reactivity of Plant Lipid Transfer Proteins (LTPs) Using Microarray in a Multicenter Study

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.     

Root character evolution and systematics in Cranichidinae, Prescottiinae and Spiranthinae (Orchidaceae, Cranichideae)

Background and Aims: Previous studies have suggested that velamen characteristicsare useful as taxonomic markers in Orchidaceae. Members of tribeCranichideae have been assigned to two velamen types constructedbased on combinations of characters such as the presence ofsecondary cell-wall thickenings and pores. However, such charactershave not been analysed on an individual basis in explicit cladisticanalyses. Methods: The micromorphology of roots of 26 species of Cranichideae wasexamined through scanning electron microscopy and light microscopy,scoring the variation and distribution of four characters: numberof velamen cell layers, velamen cell-wall thickenings, presenceand type of tilosomes, and supraendodermal spaces. The lastthree characters were analysed cladistically in combinationwith DNA sequence data of plastid trnK/matK and nuclear ribosomalinternal transcribed spacer (ITS) regions and optimized on theresulting phylogenetic tree. Key Results: Thickenings of velamen cell walls group Prescottiinae with Spiranthinae,whereas tilosomes, documented here for the first time in Cranichideae,provide an unambiguous synapomorphy for subtribe Spiranthinae.Supraendodermal spaces occur mostly in species dwelling in seasonallydry habitats and appear to have evolved three times. Conclusions: Three of the four structural characters assessed are phylogeneticallyinformative, marking monophyletic groups recovered in the combinedmolecular–morphological analysis. This study highlightsthe need for conducting character-based structural studies toovercome analytical shortcomings of the typological approach.     

Binding characteristics of bovine serum albumin encapsulated in sol-gel glasses: an alternative for protein interaction studies

Silica glasses doped with 500-700 microg of bovine serum albumin were prepared by the sol-gel method; two pH conditions (pH 5 and 7) were assayed for protein encapsulation. Both biomaterials showed a highly porous structure, with pore sizes in the range 5-28 nm. Columns packed with the ground biogels were on-line coupled to a C18 HPLC column for evaluation of the entrapped protein binding properties using propranolol. Binding capacities (at saturation) were approximately 3.7 and 7.1 microg of propranolol (drug-protein molar ratios 1.4 and 2.7) for the biogels prepared at pH 5 and 7, respectively. The significant difference indicates increased albumin denaturation upon encapsulation at pH 5. A frontal analysis study was then performed in cartridges packed with biogel prepared at pH 7 to evaluate the protein interaction with naproxen at low concentrations (相似文献   

Fe enriched biosolids as fertilizers for orange and peach trees grown in field conditions

Iron enriched biosolids (FEB) from water treatment facilities are being used as an alternative to synthetic chelates in order to improve Fe uptake. The impact of this type of products on iron nutrition is not fully understood. Plant response depends on FEB composition, soil and climatic conditions and crop response. In order to study the effectiveness of FEB as fertilisers, two field experiments have been carried out. Two different commercial formulations of FEB (unmodified u-FEB and modified m-FEB) produced as a by-product of a drinking water treatment facility in Tampa (Florida, USA) were used. An orange tree (Citrus sinensis, cv. Navelina) and a peach tree (Prunus persica cv. Sudanell) field experiments took place in different locations in Spain. Macro and micronutrients were evaluated to assess mineral status of orange and peach leaf samples. Yield and fruit size were also determined. Despite the large amount of Fe bound by the organic matter on FEB, these products were less effective than synthetic chelates to improve iron uptake. No differences were found in orange yield or size. Results show that the ferric treatments improve fruit calibre, but not yield in peaches.     

Differential effects of coinoculations with Pseudomonas jessenii PS06 (a phosphate-solubilizing bacterium) and Mesorhizobium ciceri C-2/2 strains on the growth and seed yield of chickpea under greenhouse and field conditions

In the course of a project carried out in two regions of Spain, Castilla y León and Andalucía, aiming to find useful biofertilizers for staple grain-legumes, an efficient rhizobia nodulating chickpea (termed as C-2/2) and a powerful in vitro phosphate-solubilizing bacterial strain (termed as PS06) were isolated. Analyses of their 16S rDNA sequence indicated that they belong to the bacterial species Mesorhizobium ciceri and Pseudomonas jessenii, respectively. Greenhouse and field experiments were carried out in order to test the effect of single and dual inoculations on chickpea (ecotype ILC-482) growth. Under greenhouse conditions, plants inoculated with Mesorhizobium ciceri C-2/2 alone had the highest shoot dry weight. The inoculation treatment with P. jessenii PS06 yielded a shoot dry weight 14% greater than the uninoculated control treatment, but it was not correlated with shoot P contents. However, the co-inoculation of C-2/2 with PS06 resulted in a decrease in shoot dry weight with respect to the inoculation with C-2/2 alone. Under field conditions, plants inoculated with M. ciceri C-2/2, in single or dual inoculation, produced higher nodule fresh weight, nodule number and shoot N content than the other treatments. Inoculation with P. jessenii PS06 had no significant effect on plant growth. However, the co-inoculation treatment ranked the highest in seed yield (52% greater than the uninoculated control treatment) and nodule fresh weight. These data suggest that P. jessenii PS06 can act synergistically with M. ciceri C-2/2 in promoting chickpea growth. The contrasting results obtained between greenhouse and field experiments are discussed.     

Clonal Human Fetal Ventral Mesencephalic Dopaminergic Neuron Precursors for Cell Therapy Research

A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH⁺ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experiments.     

In vitro and in vivo characterization of an interleukin‐15 antagonist peptide by metabolic stability, 99mTc‐labeling,and biological activity assays

Interleukin (IL)–15 is an inflammatory cytokine that constitutes a validated therapeutic target in some immunopathologies, including rheumatoid arthritis (RA). Previously, we identified an IL‐15 antagonist peptide named [K6T]P8, with potential therapeutic application in RA. In the current work, the metabolic stability of this peptide in synovial fluids from RA patients was studied. Moreover, [K6T]P8 peptide was labeled with ^99mTc to investigate its stability in human plasma and its biodistribution pattern in healthy rats. The biological activity of [K6T]P8 peptide and its dimer was evaluated in CTLL‐2 cells, using 3 different additives to improve the solubility of these peptides. The half‐life of [K6T]P8 in human synovial fluid was 5.88 ± 1.73 minutes, and the major chemical modifications included peptide dimerization, cysteinylation, and methionine oxidation. Radiolabeling of [K6T]P8 with ^99mTc showed a yield of approximately 99.8%. The ^99mTc‐labeled peptide was stable in a 30‐fold molar excess of cysteine and in human plasma, displaying a low affinity to plasma proteins. Preliminary biodistribution studies in healthy Wistar rats suggested a slow elimination of the peptide through the renal and hepatic pathways. Although citric acid, sucrose, and Tween 80 enhanced the solubility of [K6T]P8 peptide and its dimer, only the sucrose did not interfere with the in vitro proliferation assay used to assess their biological activity. The results here presented, reinforce nonclinical characterization of the [K6T]P8 peptide, a potential agent for the treatment of RA and other diseases associated with IL‐15 overexpression.