Engineering of caseins and modulation of their structures and interactions

β-Casein (β-CN) is a milk protein widely used in food industries because of its mild emulsifying properties due to its amphiphilicity. However, the elements determining its micellization behavior in solution and interfacial behavior at the air–water interface are not well known. In order to study how the forced dimerisation influences functional properties of β-CN, recombinant wild-type β-CN was produced and distal cysteinylated forms of recombinant β-CN were engineered. We show that 1) cysteinylated β-CN formed mainly dimers bridged by disulfide bonds; 2) the process of dimerization adds to the micellization process with temperature and is poorly reversible; 3) covalent disulfide linkage forms at the air–water interface at a lower temperature than in bulk. In conclusion, the location of the cysteinylation in the C-terminus or N-terminus or both is of importance for the properties of β-CN.     

Proteomics reveals potential biomarkers of seed vigor in sugarbeet

To unravel biomarkers of seed vigor, an important trait conditioning crop yield, a comparative proteomic study was conducted with sugarbeet seed samples of varying vigor as generated by an invigoration treatment called hydropriming and an aging treatment called controlled deterioration. Comparative proteomics revealed proteins exhibiting contrasting behavior between seed samples. Thus, 18 proteins were up-regulated during priming and down-regulated during aging and further displayed an up-regulation upon priming of the aged seeds, meaning that down-regulation of these spot volumes during aging was reversible upon subsequent priming. Also, 11 proteins exhibited the converse behavior characterized by a decrease and an increase of the spot volumes during priming and aging of the control seeds, respectively, and a decrease in the spot volumes upon priming of the aged seeds. The results underpinned the role in seed vigor of several metabolic pathways involved in lipid and starch mobilization, protein synthesis or the methyl cycle. They also corroborate previous studies suggesting that the glyoxylate enzyme isocitrate lyase, the capacity of protein synthesis and components of abscisic acid signaling pathways are likely contributors of seed vigor.     

Genetic polymorphism of the human cytochrome CYP2A13 in a French population: implication in lung cancer susceptibility

The human cytochrome CYP2A13, which is mainly expressed in the respiratory tract, has been shown to be highly efficient in vitro in the metabolism of tobacco-smoke carcinogens and procarcinogens such as 4-methylnitroso-1-(3-pyridyl)-1-butanone (NNK). In order to investigate the extent of CYP2A13 genetic polymorphism in a French Caucasian population of 102 individuals, a screening for sequence variations in the 5'-untranslated and protein encoding regions of its gene was performed using a polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) strategy. Six polymorphisms in the coding region were identified, including two rare missense mutations (C474G or Asp158Glu, G967T or Val323Leu) and one nonsense mutation (Arg101Stop). This deleterious mutation, the most frequent (5%) in our population, presumably encodes a severely truncated protein. The influence of the nonsense mutation in lung cancer susceptibility was examined by PCR-SSCP using peripheral blood DNA from 204 cases of lung cancer and 201 controls. The CYP2A13*7 allele, which harbours the C301T mutation, was present in 2.0% of controls and 3.4% of cases. However, multivariate analysis showed an elevated risk for small cell lung cancer in subjects heterozygous for the null allele (odds ratio OR=9.9; 95% confidence interval CI=1.9-52.2). This increased risk was not linked to other histological types of lung cancer.     

The Carbon Balance of Two Contrasting Mountain Forest Ecosystems in Switzerland: Similar Annual Trends,but Seasonal Differences

Net ecosystem exchange (NEE) of two contrasting mountain forest types in Switzerland was measured by eddy covariance (EC) measurements at a montane mixed forest, the Lägeren forest, over 5 years (2005–2009), and at a subalpine coniferous forest, the Seehornwald in Davos, over 12 years (1997–2009). NEE was validated against annual carbon (C) storage estimates, based on biometric and soil respiration measurements as well as soil C modeling. Three different approaches were used: (1) calculation of net ecosystem production by quantifying C pools and fluxes, (2) assessment of change in wood biomass and soil C storage (ΔC), and (3) application of biomass expansion factors. Although biometric estimates were sensitive to assumptions made for each method applied, they agreed well with measured NEE. Comparing 5 years of EC measurements available at both sites during 2005 and 2009 revealed that NEE, gross primary production (GPP), and total ecosystem respiration (TER) were larger at the Lägeren forest compared to the Davos forest, whereas soil respiration and soil C sequestration were of similar magnitudes. Both sites showed similar annual trends for NEE, GPP and TER, but different seasonal courses, due to different responses to environmental conditions (temperature, soil moisture, and radiation). Differences in the magnitude as well as in the seasonality of ecosystem CO₂ exchange could mainly be attributed to tree phenology, productivity, and carbon allocation patterns, which are combined effects of tree type (broad-leaved vs. coniferous trees) and site-specific climatic conditions. Flux differences between the two mountain sites highlight the importance of considering the role of altitude in ecological studies and modeling.     

Molecular and genetic characterization of a non-climacteric phenotype in melon reveals two loci conferring altered ethylene response in fruit

Fruit ripening and abscission are associated with an ethylene burst in several melon (Cucumis melo) genotypes. In cantaloupe as in other climacteric fruit, exogenous ethylene can prematurely induce abscission, ethylene production, and ripening. Melon genotypes without fruit abscission or without ethylene burst also exist and are, therefore, non-climacteric. In the nonabscising melon fruit PI 161375, exogenous ethylene failed to stimulate abscission, loss of firmness, ethylene production, and expression of all target genes tested. However, the PI 161375 etiolated seedlings displayed the usual ethylene-induced triple response. Genetic analysis on a population of recombinant cantaloupe Charentais x PI 161375 inbred lines in segregation for fruit abscission and ethylene production indicated that both characters are controlled by two independent loci, abscission layer (Al)-3 and Al-4. The non-climacteric phenotype in fruit tissues is attributable to ethylene insensitivity conferred by the recessive allelic forms from PI 161375. Five candidate genes (two ACO, two ACS, and ERS) that were localized on the melon genetic map did not exhibit colocalization with Al-3 or Al-4.     

Targeted apc;twist double-mutant mice: a new model of spontaneous osteosarcoma that mimics the human disease

TWIST and adenomatosis polyposis coli (APC) are critical signaling factors in normal bone development. In previous studies examining a homogeneously treated cohort of pediatric osteosarcoma patients, we reported the frequent and concurrent loss of both TWIST and APC genes. On these bases, we created a related animal model to further explore the oncogenic cooperation between these two genes. We performed intercrosses between twist-null/+ and Apc1638N/+ mice and studied their progeny. The Apc1638N/+;twistnull/+ mice developed bone abnormalities observed by macroscopic skeletal analyses and in vivo imaging. Complementary histologic, cellular, and molecular analyses were used to characterize the identified bone tumors, including cell culture and immunofluorescence of bone differentiation markers. Spontaneous localized malignant bone tumors were frequently identified in Apc1638N/+;twist-null/+ mice by in vivo imaging evaluation and histologic analyses. These tumors possessed several features similar to those observed in human localized osteosarcomas. In particular, the murine tumors presented with fibroblastic, chondroblastic, and osteoblastic osteosarcoma histologies, as well as mixtures of these subtypes. In addition, cellular analyses and bone differentiation markers detected by immunofluorescence on tumor sections reproduced most murine and human osteosarcoma characteristics. For example, the early bone differentiation marker Runx2, interacting physically with hypophosphorylated pRb, was undetectable in these murine osteosarcomas, whereas phosphorylated retinoblastoma was abundant in the osteoblastic and chondroblastic tumor subtypes. These characteristics, similar to those observed in human osteosarcomas, indicated that our animal model may be a powerful tool to further understand the development of localized osteosarcoma.     

Development of novel conductometric biosensors based on immobilised whole cell Chlorella vulgaris microalgae

A novel biosensor based on immobilised whole cell Chlorella vulgaris microalgae as a bioreceptor and interdigitated conductometric electrodes as a transducer has been developed and tested for alkaline phosphatase activity (APA) analysis. These sensors were also used for the detection of toxic compounds, namely cadmium ions, in aquatic habitats. Algae were immobilised inside bovine serum albumin (BSA) membranes cross-linked with glutaraldehyde vapours. The detection of the local conductivity variations caused by algae enzymatic reactions could be achieved. The inhibition of C. vulgaris microalgae Alkaline phosphatase activities in presence of cadmium ions was measured. These results were compared with measurements in bioassays. It finally appeared that conductometric biosensors using algae seemed more sensitive than bioassays to detect low levels of cadmium ions (the detection limit for the first experiments was 1 ppb of Cd2+). The main advantages of these alkaline phosphatase biosensors consist of their high specificity in regard to the toxic compounds they enable to detect, but also on their high stability since contrary to enzymatic biosensors, they use whole algae cells with APs on their walls.