The C-terminal fragment of the immunoproteasome PA28S (Reg alpha) as an early diagnosis and tumor-relapse biomarker: evidence from mass spectrometry profiling

This study reports on the C-terminal fragment of the 11S proteasome activator complex (PA28 or Reg alpha), a novel ovarian-specific biomarker of early and late stages of ovarian cancer (OVC) relapse, in patient biopsies after chemotherapy. A total of 179 tissue samples were analyzed: 8 stage I, 55 stage III-IV, 10 relapsed serous carcinomas, 25 mucinous carcinomas and 12 borderline and 68 benign ovarian tissue samples. This fragment was detected by MALDI mass spectrometry profiling in conjunction with a novel extraction method using hexafluoroisopropanol (1,1,1,3,3,3-hexafluoro-2-propanol; HFIP) solvents for protein solubilization and by immunohistochemistry using a specific antibody directed against the C-terminal fragment of PA28. Due to its specific cellular localization, this fragment is a suitable candidate for early OVC diagnosis, patient prognosis and follow-up during therapy and discriminating borderline cancers. Statistical analyses performed for this marker at different OVC stages reflect a prevalence of 77.66 ± 8.77 % (with a correlation coefficient value p < 0.001 of 0.601 between OVC and benign tissue). This marker presents a prevalence of 88 % in the case of tumor relapse and is detected at 80.5 % in stage I and 81.25 % ± 1.06 in stage III-IV of OVC. The correlation value for the different OVC stages is p < 0.001 of 0.998. Taken together, this report constitutes the first evidence of a novel OVC-specific marker.     

ATP hydrolysis and pristinamycin IIA inhibition of the Staphylococcus aureus Vga(A), a dual ABC protein involved in streptogramin A resistance

In Gram-positive bacteria, a large subfamily of dual ATP-binding cassette proteins confers acquired or intrinsic resistance to macrolide, lincosamide, and streptogramin antibiotics by a far from well understood mechanism. Here, we report the first biochemical characterization of one such protein, Vga(A), which is involved in streptogramin A (SgA) resistance among staphylococci. Vga(A) is composed of two nucleotide-binding domains (NBDs), separated by a charged linker, with a C-terminal extension and without identified transmembrane domains. Highly purified Vga(A) displays a strong ATPase activity (K(m) = 78 mum, V(m) = 6.8 min(-1)) that was hardly inhibited by orthovanadate. Using mutants of the conserved catalytic glutamate residues, the two NBDs of Vga(A) were shown to contribute unequally to the total ATPase activity, the mutation at NBD2 being more detrimental than the other. ATPase activity of both catalytic sites was essential for Vga(A) biological function because each single Glu mutant was unable to confer SgA resistance in the staphylococcal host. Of great interest, Vga(A) ATPase was specifically inhibited in a non-competitive manner by the SgA substrate, pristinamycin IIA (PIIA). A deletion of the last 18 amino acids of Vga(A) slightly affected the ATPase activity without modifying the PIIA inhibition values. In contrast, this deletion reduced 4-fold the levels of SgA resistance. Altogether, our results suggest a role for the C terminus in regulation of the SgA antibiotic resistance mechanism conferred by Vga(A) and demonstrate that this dual ATP-binding cassette protein interacts directly and specifically with PIIA, its cognate substrate.     

Activation mechanism of recombinant Der p 3 allergen zymogen: contribution of cysteine protease Der p 1 and effect of propeptide glycosylation

The trypsin-like protease Der p 3, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is synthesized as a zymogen, termed proDer p 3. No recombinant source of Der p 3 has been described yet, and the zymogen maturation mechanism remains to be elucidated. The Der p 3 zymogen was produced in Pichia pastoris. We demonstrated that the recombinant zymogen is glycosylated at the level of its propeptide. We showed that the activation mechanism of proDer p 3 is intermolecular and is mediated by the house dust mite cysteine protease Der p 1. The primary structure of the proDer p 3 propeptide is associated with a unique zymogen activation mechanism, which is different from those described for the trypsin-like family and relies on the house dust mite papain-like protease Der p 1. This is the first report of a recombinant source of Der p 3, with the same enzymatic activity as the natural enzyme and trypsin. Glycosylation of the propeptide was found to decrease the rate of maturation. Finally, we showed that recombinant Der p 3 is inhibited by the free modified prosequence T(P1)R.     

Expression of ActA, Ami, InlB, and Listeriolysin O in Listeria monocytogenes of Human and Food Origin

Expression of proteins involved in the adhesion of Listeria monocytogenes to mammalian cells or in the intracellular life cycle of this bacterium, including listeriolysin O (LLO), ActA, Ami, and InlB, was used to compare two populations of L. monocytogenes strains. One of the populations comprised 300 clinical strains, and the other comprised 150 food strains. All strains expressed LLO, InlB, and ActA. No polymorphism was observed for LLO and InlB. Ami was detected in 283 of 300 human strains and in 149 of 150 food strains. The strains in which Ami was not detected were serovar 4b strains. Based on the molecular weights of the proteins detected, the strains were divided into two groups with Ami (groups Ami1 [75% of the strains] and Ami2 [21%]) and into four groups with ActA (groups ActA1 [52% of the strains], ActA2 [18%], ActA3 [30%], and ActA4 [one strain isolated from food]). Logistic regression showed that food strains were more likely to belong to group ActA3 than human strains (odds ratio [OR] = 2.90; P = 1 × 10⁻⁴). Of the strains isolated from patients with non-pregnancy-related cases of listeriosis, bacteremia was predominantly associated with group Ami1 strains (OR = 1.89; P = 1 × 10⁻²) and central nervous system infections were associated with group ActA2 strains (OR = 3.04; P = 1 × 10⁻³) and group ActA3 strains (OR = 3.91; P = 1 × 10⁻³).     

Short-timescale variability of picophytoplankton abundance and cellular parameters in surface waters of the Alboran Sea (western Mediterranean)

The Alboran Sea (western Mediterranean) is characterized bya well-defined hydrological structure, the Almeria (Spain)–Oran(Algeria) geostrophic front. During the Almofront-2 cruise (November22, 1997 to January 18, 1998), high frequency sampling (     

Leaf dispersion and light partitioning in three-dimensionally digitized tall fescue–white clover mixtures

A three‐dimensional digitizing method was used to assess the canopy structure of six Festuca arundinacea (FA)–Trifolium repens (TR) mixtures during the installation stage. Virtual canopy images were synthesized and used to derive light interception and partitioning between species. Computations from images were compared with a simple light model based on Beer’s law, in order to analyse within‐ and between‐species foliage dispersion. The total leaf area index of the mixtures ranged from 0·6 to 4·5. The fraction of FA foliage overtopping TR was 9–30%. The mean inclination of FA and TR was 66 and 57°, respectively. Within‐species dispersion parameters of FA and TR were about 0·8 and 1·0, namely clumped and random foliage dispersion, respectively. Although FA was sown in rows, between‐species dispersion was random. Lower leaf inclination and lesser clumping in TR compensated foliage overtopping by FA, so that light partitioning between FA and TR (about 80 and 20%, respectively) was similar to the species contribution to total canopy foliage. Since between‐species dispersion was random, a simple two‐layer light model based on Beer’s law provided correct estimations of light partitioning (RMSE = 0·05), although light interception by FA was slightly overestimated because of its clumped dispersion.     

Expression of a Recombinant Toxoplasma gondii ROP2 Fragment as a Fusion Protein in Bacteria Circumvents Insolubility and Proteolytic Degradation

A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii rhoptry protein ROP2 (recROP2_t, residues 196–464) was expressed in Escherichia coli. This recombinant fragment was produced at low concentration and in a highly insoluble form. By contrast, the level of recROP2_t production was drastically greater when the same coding sequence was fused to the C-terminus of thioredoxin (TRX) or to the maltose-binding protein (MBP) gene. While both fusion proteins were found to be mainly insoluble, solubilization could be achieved without significant degradation. MBP was more efficient than TRX in increasing the recovery of soluble protein with more than 10% of total MBP–recROP2_t being readily expressed in a soluble form. Moreover, the insoluble form of MBP–recROP2_t could be correctly refolded with a recovery of more than 80%. Both forms of MBP–recROP2_t were purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX–recROP2_t promoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by gel filtration. Subsequent proteolytic cleavages of purified TRX–recROP2_t and of MBP–recROP2_t led respectively to the complete degradation or to the truncation of the recROP2_t moiety. However, recROP2_t, despite the presence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Finally, both fusion proteins were able to induce specific humoral and cell-mediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondii proteins which are difficult to produce because of insolubility and degradation.     

Traits émotionnels, intelligence émotionnelle: intÉrêt de ces concepts et Étude de leurs interrelations

Studies on personality traits conducted during the past decade indicate that there is a set of emotion-related traits on which individuals differ. Moreover, other studies showed that there are some abilities related to the processing of both emotions and emotional information, referred to as the concept of emotional intelligence. The authors of the article evaluated one hundred adults (ranging in age from 20–50) using the French version of a series of scales to measure stable emotionrelated traits and emotional intelligence. The results showed that these instruments provide accurate internal consistency and reliability, and that there are significant relationships between the individual emotional characteristics observed. A factorial analysis conducted with varimax rotation underlined five primary factors identified as: Clear-sightedness of emotions, Emotional richness, Identification, Understanding and Emotional control. The average emotional profiles based on these five primary factors depend on gender and age. In particular, the female and the oldest participants showed a greater emotional richness. The examination of these five factors should lead to a better understanding of the relationships which exist between these emotional characteristics and cognitive performances, and their involvement in clinical syndromes.     

Seasonal and spatial variability of virio-, bacterio-, and picophytoplanktonic abundances in three peri-alpine lakes

Flow cytometry (FCM) was used to assess microbial community abundances and patterns in three natural, large and deep peri-alpine hydrosystems, i.e., lakes Annecy (oligotrophic), Bourget, and Geneva (mesotrophic). Picocyanobacteria, small eukaryotic autotrophs, heterotrophic prokaryotes, and viruses were studied in the 0–50 m surface layers to highlight the impact of both physical and chemical parameters as well as possible biotic interactions on the functioning of microbial communities. Some specificities were recorded according to the trophic status of each ecosystem such as the higher number of viruses and heterotrophic bacteria in mesotrophic environments (i.e., Lakes Geneva and Bourget) or the higher abundance of picocyanobacteria in the oligotrophic Lake Annecy. However, both seasonal (temperature) and spatial (depth) variations were comparatively more important than the trophic status in driving the microbial communities’ abundances in these three lakes, as revealed by principal component analysis (PCA). A strong viral termination of the heterotrophic bacterial blooms could be observed in autumn for each lake, in parallel to the mixing of the upper lit layers. As virus to bacteria ratio (VBR) was indeed very high at this period with values varying between 87 and 114, such important relationships between viruses and bacteria were likely. The magnitudes of seasonal variations in VBR, with the highest values ever reported so far, were largely greater than the magnitude of theoretical variations due to the trophic status, suggesting also a strong seasonality in virioplankton production associated to prokaryotic dynamics. FCM analyses allowed discriminating several viral groups. Virus-Like Particles group 1 (VLP1) and group 2 (VLP2) were always observed and significantly correlated to bacteria for the former and chlorophyll a and picocyanobacteria for the latter, suggesting that most of VLP1 and VLP2 could be bacteriophages and cyanophages, respectively. On the basis of these results, new ways of investigation emerge concerning the study of relationships between specific picoplanktonic groups; and overall these results provide new evidence of the necessity to consider further viruses for a better understanding of lake plankton ecology. Handling editor: Luigi Naselli-Flores