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.     

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.     

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.     

Acivicin induces apoptosis independently of gamma-glutamyltranspeptidase activity

To elucidate the molecular mechanism(s) involved in the TRAIL-induced apoptosis sensitivity, we conducted the following experiments utilizing TRAIL-sensitive and -resistant glioma cells. We examined the expression of TRAIL receptors mRNA, but no significant differences were detected in those cells. TRAIL-resistant cells were sensitized to TRAIL-induced apoptosis by staurosporine pretreatment and preferentially expressed PKCepsilon. Since several lines of evidence suggest that PKC may play a protective role for apoptosis, we analyzed the involvement of PKCepsilon in TRAIL-induced apoptosis by an adenovirus vector expression system. We found that TRAIL susceptibility was augmented by the expression of a dominant negative PKCepsilon in TRAIL-resistant cells. Conversely, PKCepsilon introduction in TRAIL-sensitive cells resulted in the reduction of TRAIL-induced apoptosis. Taken together, these data suggest that PKCepsilon may be a regulator of susceptibility to TRAIL-induced apoptosis in gliomas and probably other malignancies.     

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.     

Comparative effects of antiepileptics on the alkaline phosphatase and gamma glutamyltransferase activities in plasma and leukocytes]

Alkaline phosphatase and gamma glutamyltransferase activities have been compared in plasma and leukocytes from presumably healthy subjects and from epileptic patients under treatment with different antiepileptic drugs. Plasma enzyme activities are always increased by antiepileptic treatment, leukocytic enzyme activities are increased only for some patients. No relation has been observed between the variations in any of the two enzyme activities and plasma level of anticonvulsant drugs.     

Differential time-course of induction of rat liver gamma-glutamyltransferase and drug-metabolizing enzymes in the endoplasmic reticulum, Golgi and plasma membranes after a single phenobarbital injection. Evaluation of protein variations by two-dimensional electrophoresis

This study was conducted to follow as a function of time the activity of gamma-glutamyltransferase in the various membranes of rat liver cells after a single dose of phenobarbital (PB) (75 mg kg-1 body weight). Gamma-glutamyltransferase induction was maximal 24 h after PB treatment in both the rough endoplasmic reticulum and the plasma membranes. This pattern of induction differed from that of some drug metabolizing enzymes. While total cytochrome P-450 content was enhanced mainly in endoplasmic reticulum until 48 h after PB treatment, UDP-glucuronosyltransferase activity was not greatly altered by PB under the same conditions. The comparison of two-dimensional electrophoretic polypeptide profiles of each subcellular membrane isolated from control and phenobarbital-treated rats revealed important variations induced by PB. In plasma membranes, the heaviest subunit (apparent Mr = 60 x 10(3)) of hepatic gamma-glutamyltransferase was provisionally identified as a collection of polypeptide which differ only by their pI. The concentration of these polypeptides was smaller in the endoplasmic reticulum where they were of lower apparent molecular mass. This suggests that the gamma-glutamyltransferase precursor is already processed at the level of the endoplasmic reticulum but it is still not completely mature or glycosylated. Five days of continuous PB treatment induced by appearance of new gamma-glutamyltransferase isoforms in plasma membranes. We demonstrate that after a single injection of PB, gamma-glutamyltransferase activity increases simultaneously with some drug-metabolizing enzymes, such as total cytochrome P-450 but not with others, such as UDP-glucuronosyltransferases.     

Early traces of life investigations in drilling Archean hydrothermal and sedimentary rocks of the Pilbara Craton,Western Australia and Barberton Greenstone Belt,South Africa

The Pilbara Craton of Western Australia and the Barberton Greenstone Belt of the Kaapvaal Craton, South Africa, contain some of the oldest and best preserved Archaean rocks and microfossils in the world. Two stratigraphic horizons in the Pilbara Craton were drilled as part of a collaborative effort between France and Australia (the Pilbara Drilling Project) during August 2004, including the 3481 Ma Dresser Formation (Warrawoona Group) and 2724 Ma Tumbiana Formation (Fortescue Group). A new diamond drill hole was cored in August 2008 through part of the ～3250 Ma Fig Tree Group in the Barberton Greenstone Belt as part of a joint project between France and South Africa. These pristine diamond drill cores present a unique opportunity to constrain the chemistry of the earliest ocean, the composition of the atmosphere, and the settings and types of microbial ecosystems spanning the Archean Eon. These drill core samples can also provide new clues on the earliest metabolic pathways.