Genomic organisation of the <Emphasis Type="Italic">Mal d 1</Emphasis> gene cluster on linkage group 16 in apple

European populations exhibit progressive sensitisation to food allergens, and apples are one of the foods for which sensitisation is observed most frequently. Apple cultivars vary greatly in their allergenic characteristics, and a better understanding of the genetic basis of low allergenicity may therefore allow allergic individuals to increase their fruit intake. Mal d 1 is considered to be a major apple allergen, and this protein is encoded by the most complex allergen gene family. Not all Mal d 1 members are likely to be involved in allergenicity. Therefore, additional knowledge about the existence and characteristics of the different Mal d 1 genes is required. In the present study, we investigated the genomic organisation of the Mal d 1 gene cluster in linkage group 16 of apple through the sequencing of two bacterial artificial chromosome clones. The results provided new information on the composition of this family with respect to the number and orientation of functional and pseudogenes and their physical distances. The results were compared with the apple and peach genome sequences that have recently been made available. A broad analysis of the whole apple genome revealed the presence of new genes in this family, and a complete list of the observed Mal d 1 genes is supplied. Thus, this study provides an important contribution towards a better understanding of the genetics of the Mal d 1 family and establishes the basis for further research on allelic diversity among cultivars in relation to variation in allergenicity.     

Genotyping of Cryptosporidium Isolates from Chamelea gallina Clams in Italy

Chamelea gallina clams collected from the mouths of rivers along the Adriatic Sea (central Italy) were found to harbor Cryptosporidium parvum (genotype 2), which is the lineage involved in zoonotic transmission. The clams were collected from the mouths of rivers near whose banks ruminants are brought to graze. This paper reports the environmental spread of C. parvum in Italy and highlights the fact that genotyping of seaborne Cryptosporidium isolates is a powerful tool with which to investigate the transmission patterns and epidemiology of this microorganism.     

Sequence-selective peptide detection by small synthetic chemosensors selected from an encoded combinatorial chemosensor library.

Synthetic chemosensors hold great potential in many diagnostic applications. In this study, we describe the design and preparation of the first encoded combinatorial library of chemosensors for tripeptides. Subsequent screening of the library resulted in the discovery of novel chemosensors able to distinguish between random tripeptides.     

Chemistry and in vitro mutagenicity of 2-aminofluorene derivatives

A study on the relationship between mutagenic activity and chemical reactivity of a series of 2-fluorenylamino and hydroxylamino derivatives has been carried out by assaying their ability to revert the Salmonella typhimurium strain TA98. The mutagenic potency of the fluorenamides increased with increasing availability of the amidic hydrogen for abstraction and tertiary amides were quite inactive. N-Hydroxy and N-acyloxy derivatives were directly mutagenic and increased their mutagenic activity after metabolic conversion by liver S9. N-Hydroxy-2-benzoylaminofluorene, inactive without S9, after activation was the most mutagenic. Of a pair of N-acyloxy-derivatives, N-benzoyloxy-2-acetylaminofluorene, which undergoes rearrangement of the benzoyloxy group from nitrogen to ring carbons even at room temperature, was less potent than N-acetyloxy-2-acetylamino-fluorene whose rearrangement occurs at higher temperatures. Corresponding C-1 and C-3 benzoyloxy and acetyloxy derivatives were found ineffective in this assay in agreement with previous reports on the hydroxy series. N-Chloro-2-amino-(or acetylamino)fluorene were found more active than the corresponding N-hydroxy analogs in the presence of S9, thus suggesting an alternate pathway for activation, likely a direct conversion to electrophilic species. Furthermore, in contrast with inactivity of ring hydroxy and acyloxy derivatives, 3-chloro-2-acetylaminofluorene retained mutagenic activity. Finally 2,2'-azoxyfluorene, the ultimated oxidation product of N-hydroxyaminofluorene, tested in vitro and in vivo experiments, was found completely inactive.     

Dynamic properties of monomeric insect erythrocruorin III from Chironomus thummi-thummi: relationships between structural flexibility and functional complexity.

We have investigated the kinetics of geminate carbon monoxide binding to the monomeric component III of Chironomus thummi-thummi erythrocruorin, a protein that undergoes pH-induced conformational changes linked to a pronounced Bohr effect. Measurements were performed from cryogenic temperatures to room temperature in 75% glycerol and either 0.1 M potassium phosphate (pH 7) or 0.1 potassium borate (pH 9) after nanosecond laser photolysis. The distributions of the low temperature activation enthalpy g(H) for geminate ligand binding derived from the kinetic traces are quite narrow and are influenced by temperature both below and above approximately 170 K, the glass transition temperature. The thermal evolution of the CO binding kinetics between approximately 50 K and approximately 170 K indicates the presence of some degree of structural relaxation, even in this temperature range. Above approximately 220 K the width of the g(H) progressively decreases, and at 280 K geminate CO binding becomes exponential in time. Based on a comparison with analogous investigations of the homodimeric hemoglobin from Scapharca inaequivalvis, we propose a link between dynamic properties and functional complexity.     

Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts.

Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n/CH3 peak areas R=1. 56+/-0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. Involvement of phosphatidylcholine hydrolysis in ML generation was demonstrated by selective inhibition of endogenous phospholipase C (PC-plc) or by exposure to bacterial PC-plc. This study indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles (possibly in mutual dynamic exchange) may contribute (although to a different extent) to ML signals; and (2) high levels of ras-transfection either inhibit ML formation or facilitate their extrusion from the cell.     

Interaction of trout hemoglobin with H2O2: a chemiluminescence study

Erythrocytes from trout Salmo irideus are characterized by four different hemoglobin components (HbI, HbII, HbIII and HbIV), HbI and HbIV being predominant. In this study we describe the interaction between trout hemoglobin (HbI and HbIV) and H₂O₂ using a chemiluminescence assay. Our data show that the reaction of hemoglobins with H₂O₂ produces a time-limited and significant increase of chemiluminescence signal. The half-life of the decay of this chemiluminescence signal was characteristic for each type of hemoglobin used. These results indicate the formation of excited molecules related to the interaction between trout hemoglobin and H₂O₂. © 1997 John Wiley & Sons, Ltd.     

Modulation of the TGF-β1-induced epithelial to mesenchymal transition (EMT) mediated by P1 and P2 purine receptors in MDCK cells

Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-β1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-β1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-β1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation of A2A receptor reduced the overexpression of the EMT-related markers, mainly through the cAMP-dependent PKA pathway, as confirmed by cell pre-treatment with Myr-PKI. Both A1 and P2Y1 receptor stimulation exacerbated the TGF-β1-driven effects, which were reduced by cell pre-treatment with the MAPK inhibitor PD98059, according to the increased ERK1/2 phosphorylation upon receptor activation. The effects induced by P2Y11 receptor activation were oppositely modulated by PKA or MAPK inhibition, in line with the dual nature of the Gs- and Gq-coupled receptor. Differently, P2X7 receptor induced, per se, similar and not additive effects compared to TGF-β1, after prolonged cell exposure to BzATP. These results suggest a putative role of purine receptors as target for anti-fibrotic agents.