Investigation at residue level of the early steps during the assembly of two proteins into supramolecular objects

Understanding the driving forces governing protein assembly requires the characterization of interactions at molecular level. We focus on two homologous oppositely charged proteins, lysozyme and α-lactalbumin, which can assemble into microspheres. The assembly early steps were characterized through the identification of interacting surfaces monitored at residue level by NMR chemical shift perturbations by titrating one (15)N-labeled protein with its unlabeled partner. While α-lactalbumin has a narrow interacting site, lysozyme has interacting sites scattered on a broad surface. The further assembly of these rather unspecific heterodimers into tetramers leads to the establishment of well-defined interaction sites. Within the tetramers, most of the electrostatic charge patches on the protein surfaces are shielded. Then, hydrophobic interactions, which are possible because α-lactalbumin is in a partially folded state, become preponderant, leading to the formation of larger oligomers. This approach will be particularly useful for rationalizing the design of protein assemblies as nanoscale devices.     

Active metabolism of arachidonic acid by Kaposi sarcoma cells cultured from lung biopsies (KS-3); identification by HPLC and MS/MS of the predominant metaboilte secreted as the 11,12-epoxy-eicosatrienoic acid

The development of long-term culture of AIDS-KS cells has allowed us to investigate further a possible vascular origin of Kaposi sarcoma. Taking into account the relative specificity of arachidonic acid (AA) metabolism according to cell type, the AA ‘cascade’ was analyzed in cultured KS-3 cells established from lung biopsies and compared to human umbilical venous endothelial (H-UVE) cells and human myometrial smooth muscle (H-MSM) cells, under basal conditions and after stimulation with vasoactive agents such as histamine or thrombin. Considering strictly the ‘prostaglandin’ profile given by RIAs, the metabolism of AA was closer, whilst not identical, to H-UVE than to H-MSM cells. However, evaluation of all the eicosanoids released from [³H]AA labeled KS-3 cells revealed that the predominant metabolite was not prostacyclin (PGI₂), as suggested from PG RIAs, but an epoxy-eicosatrienoic acid (EET), identified as the 11, 12 isomer by HPLC and MS/MS. The synthesis of this EET is probably cytochrome P-450 monooxygenase dependent. Its potential role in the development of the KS tumor cells is under investigation.     

The expression of Quox 1, a homeodomain-containing protein, in sympathetic ganglion cells is regulated in vitro by growth factors

Quox 1, a quail homeobox gene, is the first vertebrate Antp-type homeobox gene to be described that is expressed in the forebrain. We have already shown that the Quox 1 protein is specifically expressed in post-mitotic sensory neurons. A subpopulation of sympathetic ganglion cells was also found to be labelled by anti-Quox 1 in vitro, but it is not clear whether this protein is expressed in sympathetic ganglion cells in vivo and, if so, the conditions which regulate its expression in vitro. In the present study, we used immunocytochemistry to find out whether Quox 1 expression in sympathetic ganglion cells in vitro is regulated by environmental signals. We found that several peptide growth factors can regulate Quox 1 expression in cultured sympathetic ganglion cells, and that they do so at physiological concentration and in a variety of ways. Basic fibroblast growth factor (FGF-2) induces Quox 1 protein expression, whereas insulin and human insulin-like growth factor-I (IGF-I) down-regulate Quox 1 expression.     

Kinetics of fibril formation of bovine kappa-casein indicate a conformational rearrangement as a critical step in the process

S-carboxymethylated (SCM) κ-casein forms in vitro fibrils that display several characteristics of amyloid fibrils, although the protein is unrelated to amyloid diseases. In order to get insight into the processes that prevent the formation of amyloid fibrils made of κ-caseins in milk, we have characterized in detail the reaction and the roles of its possible effectors: glycosylation and other caseins. Given that native κ-casein occurs as a heterogeneous mixture of carbohydrate-free and carbohydrate-containing chains, kinetics of fibril formation were performed on purified glycosylated and unglycosylated SCM κ-caseins using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and Fourier transform infrared spectroscopy for morphological and structural analyses. Both unglycosylated and glycosylated SCM κ-caseins have the ability to fibrillate. Kinetic data indicate that the fibril formation rate increases with SCM κ-casein concentration but reaches a plateau at high concentrations, for both the unglycosylated and glycosylated forms. Therefore, a conformational rearrangement is the rate-limiting step in fibril growth of SCM κ-casein. Transmission electron microscopy images indicate the presence of 10- to 12-nm spherical particles prior to the appearance of amyloid structure. Fourier transform infrared spectroscopy spectra reveal a conformational change within these micellar aggregates during the fibrillation. Fibrils are helical ribbons with a pitch of about 120-130 nm and a width of 10-12 nm. Taken together, these findings suggest a model of aggregation during which the SCM κ-casein monomer is in rapid equilibrium with a micellar aggregate that subsequently undergoes a conformational rearrangement into a more organized species. These micelles assemble and this leads to the growing of amyloid fibrils. Addition of α_s1-and β-caseins decreases the growth rate of fibrils. Their main effect was on the elongation rate, which became close to that of the limiting conformation change, leading to the appearance of a lag phase at the beginning of the kinetics.     

Decomposition of 13C-labelled standard plant material in a latitudinal transect of European coniferous forests: Differential impact of climate on the decomposition of soil organic matter compartments

¹³C labelled plant material was incubated in situ over 2 to 3 years in 8 conifer forest soils located on acid and limestone parent material along a north-south climatic transect from boreal to dry Mediterranean regions in western Europe. The objectives of the experiment were to evaluate the effects of climate and the soil environment on decomposition and soil organic matter dynamics. Changes in climate were simulated using a north-to-south cascade procedure involving the relocation of labelled soil columns to the next warmer site along the transect.Double exponential, decay-rate functions (for labile and recalcitrant SOM compartments) vs time showed that the thermosensitivity of microbial processes depended on the latitude from which the soil was translocated. Cumulative response functions for air temperature, and for combined temperature and moisture were used as independent variables in first order kinetic models fitted to the decomposition data. In the situations where climatic response functions explained most of the variations in decomposition rates when the soils were translocated, the climate optimised decomposition rates for the local and the translocated soil should be similar. Differences between these two rates indicated that there was either no single climatic response function for one or both compartments, and/or other edaphic factors influenced the translocation effect. The most northern boreal soil showed a high thermosensitivity for recalcitrant organic matter compartment, whereas the labile fraction was less sensitive to climate changes for soils from more southern locations. Hence there was no single climatic function which describe the decay rates for all compartments. At the end of the incubation period it was found that the heat sum to achieve the same carbon losses was lower for soils in the north of the transect than in the south. In the long term, therefore, for a given heat input, decomposition rates would show larger increases in boreal northern sites than in warm temperate regions.The changes in climate produced by soil translocation were more clearly reflected by decomposition rates in the acid soils than for calcareous soils. This indicates that the physicochemical environment can have important differential effects on microbial decomposition of the labile and recalcitrant components of SOM.     

Do tree species characteristics influence soil respiration in tropical forests? A test based on 16 tree species planted in monospecific plots

The high spatial variability of soil respiration in tropical rainforests is well evaluated, but influences of biotic factors are not clearly understood. This study underlines the influence of tree species characteristics on soil respiration across a 16-monospecific plot design in a tropical plantation of French Guiana. A large variability of soil CO₂ fluxes was observed among plots (i.e. 2.8 to 6.8 μmol m^?2 s^?1) with the ranking being constant across seasons. There were no significant relationships between soil respiration and soil moisture or soil temperature, neither spatially, nor seasonally. The variability of soil respiration was mainly explained by quantitative factors such as leaf litterfall and basal area. Surprisingly, no significant relationship was observed between soil respiration and root biomass. However, the influence of substrate quality was revealed by a strong relationship between soil respiration and litterfall P (and litterfall N, to a lesser extent).     

Effects on the cytoskeleton of a new inducer of the neuroblastoma morphological differentiation

1-Methyl cyclohexane carboxylic acid (CCA), a new inducer of neuroblastoma morphological differentiation, was found to stimulate the synthesis of vimentin in neuroblastoma cultures. Synthesis of actin and isotubulins was also modified, thus implying a more general effect on the cytoskeleton. Most of these variations are observed in culture conditions allowing interactions between the cell membrane and a solid substrate.