One-electron oxidation of beta-amyloid peptide: sequence modulation of reactivity

Amyloid beta peptide (Abeta) is a 39 to 43 amino-acid-long peptide implicated in Alzheimer's disease. One of its mechanisms of toxicity is related to its redox properties. Therefore we studied its one electron oxidation using azide free radicals produced in gamma and pulse radiolysis, and compared the results with those obtained with the reverse sequence Abeta(40-1). HPLC analysis combined with absorption, fluorescence, Raman spectroscopy, and MALDI-TOF MS were used for product identification. Met35 was shown to be the target in Abeta(1-40); oxidation leads to a major compound that is Abeta with methionine sulfoxide. Similarly, oxidation of fragment Abeta(29-40) also leads to methionine sulfoxide. For Abeta(40-1), Met35 is not reactive and Tyr10 is the target of azide radicals. The major products are peptide dimer linked by dityrosine and trimer. The lowering of the one-electron reduction potential of the MetS+/Met couple, which was proposed, is in agreement with our findings. To our knowledge, this is the first time that such a drastic effect of the primary sequence is observed in a small peptide. In addition, it is also the first experimental demonstration of the sensitivity of the one-electron reduction potential of methionine on neighboring groups.     

Hormones, IgG and lactose changes around parturition in plasma, and colostrum or saliva of multiparous sows

Blood, colostrum and saliva samples were serially taken from 6 multiparous sows from day 109 of gestation until day 3 postpartum. Plasma was assayed for oestradiol-17beta (E2), progesterone (P4), prolactin (PRL), cortisol, immunoglobulin G (IgG) and lactose. Colostrum was assayed for E2, P4, IgG and lactose. Lactoserum, obtained after ultra centrifugation of colostrum, was assayed for PRL. Saliva was assayed for cortisol. Time-related variations in hormone, IgG and lactose concentrations measured in plasma were parallel to those measured in colostrum, lactoserum or saliva. However, the concentrations were higher in colostrum or lactoserum and lower in saliva than in plasma. Ratios of concentrations of cortisol in saliva and PRL in lactoserum over those in plasma did not vary with time and averaged 0.2 and 1.6, respectively. Conversely, the ratios of concentrations of E2 and P4 in colostrum over those in plasma varied with time (P < 0.05) but were quite constant before the end of parturition, averaging 2.7 and 3.6, respectively. The ratios of concentrations of IgG and lactose in colostrum over those in plasma also varied with time (P < 0.05). The concentrations of hormones in plasma on the one hand and in colostrum, lactoserum or saliva on the other hand were significantly correlated but correlations varied with time (PRL across periods: r = 0.31; cortisol across periods: r = 0.60; E2 during parturition: r = 0.83; P4 before parturition: r = 0.82; P4 during parturition: r = 0.67). The present results indicate that around parturition, assays of hormones in colostrum or saliva can be used to study the hormonal status of sows. Furthermore, variations in colostrum and plasma concentrations of IgG and lactose are good indicators of the transition from colostrum to milk synthesis.     

Use of antisense RNA to modify the composition of cellulosomes produced by Clostridium cellulolyticum

The enzymatic composition of the cellulosomes produced by Clostridium cellulolyticum was modified by inhibiting the synthesis of Cel48F that is the major cellulase of the cellulosomes. The strain ATCC 35319 (pSOSasrF) was developed to over-produce a 469 nucleotide-long antisense-RNA (asRNA) directed against the ribosome-binding site region and the beginning of the coding region of the cel48F mRNAs. The cellulolytic system secreted by the asRNA-producing strain showed a markedly lower amount of Cel48F, compared to the control strain transformed with the empty plasmid (pSOSzero). This was correlated with a 30% decrease of the specific activity of the cellulolytic system on Avicel cellulose, indicating that Cel48F plays an important role in the recalcitrant cellulose degradation. However, only minor effects were observed on the growth parameters on cellulose. In both transformant strains, cellulosome production was found to be reduced and two unknown proteins (P105 and P98) appeared as major components of their cellulolytic systems. These proteins did not contain any dockerin domain and were shown to be not included into the cellulosomes; they are expected to participate to the non-cellulosomal cellulolytic system of C. cellulolyticum.     

Epimerization of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium baratii isolated from human feces

Several H2-producing fermentative anaerobic bacteria including Clostridium, Klebsiella and Fusobacteria degraded octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (36 microM) to formaldehyde (HCHO) and nitrous oxide (N2O) with rates ranging from 5 to 190 nmol h(-1)g [dry weight] of cells(-1). Among these strains, C. bifermentans strain HAW-1 grew and transformed HMX rapidly with the detection of the two key intermediates the mononitroso product and methylenedinitramine. Its cellular extract alone did not seem to degrade HMX appreciably, but degraded much faster in the presence of H2, NADH or NADPH. The disappearance of HMX was concurrent with the release of nitrite without the formation of the nitroso derivative(s). Results suggest that two types of enzymes were involved in HMX metabolism: one for denitration and the second for reduction to the nitroso derivative(s).     

Arabidopsis formin AtFH6 is a plasma membrane-associated protein upregulated in giant cells induced by parasitic nematodes

Plant-parasitic nematodes Meloidogyne spp induce an elaborate permanent feeding site characterized by the redifferentiation of root cells into multinucleate and hypertrophied giant cells. We have isolated by a promoter trap strategy an Arabidopsis thaliana formin gene, AtFH6, which is upregulated during giant cell formation. Formins are actin-nucleating proteins that stimulate de novo polymerization of actin filaments. We show here that three type-I formins were upregulated in giant cells and that the AtFH6 protein was anchored to the plasma membrane and uniformly distributed. Suppression of the budding defect of the Saccharomyces cerevisiae bni1Delta bnr1Delta mutant showed that AtFH6 regulates polarized growth by controlling the assembly of actin cables. Our results suggest that AtFH6 might be involved in the isotropic growth of hypertrophied feeding cells via the reorganization of the actin cytoskeleton. The actin cables would serve as tracks for vesicle trafficking needed for extensive plasma membrane and cell wall biogenesis. Therefore, determining how plant parasitic nematodes modify root cells into giant cells represents an attractive system to identify genes that regulate cell growth and morphogenesis.     

The structure of Acidithiobacillus ferrooxidans c(4)-cytochrome: a model for complex-induced electron transfer tuning

The study of electron transfer between the copper protein rusticyanin (RCy) and the c(4)-cytochrome CYC(41) of the acidophilic bacterium Acidithiobacillus ferrooxidans has evidenced a remarkable decrease of RCy's redox potential upon complex formation. The structure of the CYC(41) obtained at 2.2 A resolution highlighted a specific glutamate residue (E121) involved in zinc binding as potentially playing a central role in this effect, required for the electron transfer to occur. EPR and stopped-flow experiments confirmed the strong inhibitory effect of divalent cations on CYC(41):RCy complex formation. A docking analysis of the CYC(41) and RCy structure allows us to propose a detailed model for the complex-induced tuning of electron transfer in agreement with our experimental data, which could be representative of other copper proteins involved in electron transfer.