Phycobilisome linker proteins are phosphorylated in Synechocystis sp. PCC 6803

The controversial issue of protein phosphorylation from the photosynthetic apparatus of Synechocystis sp. PCC 6803 has been reinvestigated using new detection tools that include various immunological and in vivo labeling approaches. The set of phosphoproteins detected with these methods includes ferredoxin-NADPH reductase and the linker proteins of the phycobilisome antenna. Using mutants that lack a specific set of linker proteins and are affected in phycobilisome assembly, we show that the phosphoproteins from the phycobilisomes correspond to the membrane, rod, and rod-core linkers. These proteins are in a phosphorylated state within the assembled phycobilisomes. Their dephosphorylation requires partial disassembly of the phycobilisomes and further contributes to their complete disassembly in vitro. In vivo we observed linker dephosphorylation upon long-term exposure to higher light intensities and under nitrogen limitation, two conditions that lead to remodeling and turnover of phycobilisomes. We conclude that this phosphorylation process is instrumental in the regulation of assembly/disassembly of phycobilisomes and should participate in signaling for their proteolytic cleavage and degradation.     

Plasmodium falciparum: production of human antibodies specific for the MSP-3 protein in the Hu-SPL-SCID mouse

Human immunity against Plasmodium falciparum malaria is mediated by IgG antibodies. One of the major targets of protective antibodies is the MSP-3 protein. Anti-MSP-3 human monoclonal antibodies could therefore be valuable for passive immunotherapy, particularly of drug resistant malaria. Human monoclonal antibodies were previously produced in the Hu-SPL-SCID model reconstituted with human splenocytes, immunized by highly immunogenic neo-antigen or a recall antigen. We report here that this model can also be successfully employed to induce human antibody-secreting cells specific of low immunogenicity neo-antigens, such as MSP-3. These cells represent a new and valuable source of human monoclonal anti-malaria antibodies.     

Caveolin-1 deficiency stimulates neointima formation during vascular injury

Neointima formation is a process characterized by smooth muscle cell (SMC) proliferation and extracellular matrix deposition in the vascular intimal layer. Here, we critically evaluate the role of caveolin-1 (Cav-1) in the pathogenesis of neointima formation. Cav-1 and caveolae organelles are particularly abundant in SMCs, where they are thought to function in membrane trafficking and signal transduction events. To directly evaluate the role of Cav-1 in the pathogenesis of neointimal lesions, we used Cav-1-deficient (Cav-1 -/-) mice as a model system. The right common carotid artery of wild-type and Cav-1 -/- mice was ligated just proximal to its bifurcation. Specimens were then harvested 4-weeks postligation and processed for morphometric and immunohistochemical analyses. The carotids of Cav-1 -/- mice showed significantly more intimal hyperplasia with subtotal luminal obstruction, as compared to wild-type mice. These neointimal lesions consisted mainly of SMCs. Mechanistically, neointimal lesions derived from Cav-1 -/- mice exhibited higher levels of phospho-p42/44 MAP kinase and cyclin D1 immunostaining, consistent with the idea that Cav-1 functions as a negative regulator of signal transduction. A significant increase in phospho-Rb (Ser780) immunostaining was also observed, in line with the upregulation of cyclin D1. In conclusion, using a carotid artery blood-flow cessation model, we show that genetic ablation of Cav-1 in mice stimulates SMC proliferation (neointimal hyperplasia), with concomitant activation of the p42/44 MAP kinase cascade and upregulation of cyclin D1. Importantly, our current study is the first to investigate the role of Cav-1 in SMC proliferation in the vascular system using Cav-1 -/- mice.     

Characterization of the reactivity determinants of a novel hairpin substrate of yeast RNase III

RNase III enzymes form a conserved family of proteins that specifically cleave double-stranded (dsRNA). These proteins are involved in a variety of cellular functions, including the processing of many non-coding RNAs, mRNA decay, and RNA interference. Yeast RNase III (Rnt1p) selects its substrate by recognizing the structure generated by a conserved NGNN tetraloop (G2-loop). Mutations of the invariant guanosine stringently inhibit binding and cleavage of all known Rnt1p substrates. Surprisingly, we have found that the 5' end of small nucleolar RNA 48 is processed by Rnt1p in the absence of a G2-loop. Instead, biochemical and structural analyses revealed that cleavage, in this case, is directed by a hairpin capped with an AAGU tetraloop, with a preferred adenosine in the first position (A1-loop). Chemical probing indicated that A1-loops adopt a distinct structure that varies at the 3' end where Rnt1p interacts with G2-loops. Consistently, chemical footprinting and chemical interference assays indicate that Rnt1p binds to G2 and A1-loops using different sets of nucleotides. Also, cleavage and binding assays showed that the N-terminal domain of Rnt1p aids selection of A1-capped hairpins. Together, the results suggest that Rnt1p recognizes at least two distinct classes of tetraloops using flexible protein RNA interactions. This underscores the capacity of double-stranded RNA binding proteins to use several recognition motifs for substrate identification.     

De-ubiquitinating proteases USP2a and USP2c cause apoptosis by stabilising RIP1

Dynamic ubiquitination impacts on the degradation of proteins by the proteasome as well as on their effects as signalling factors. Of the many cellular responses that are regulated by changes in ubiquitination, apoptosis has garnered special attention. We have found that USP2a and USP2c, two isoforms of the ubiquitin-specific protease USP2, cause cell death upon ectopic expression. We show that both USP2 isoforms can control the ubiquitination status of many proteins but from a panel of potential targets only the protein level of RIP1 was increased by these enzymes. This effect is responsible for the activity of USP2a and USP2c to cause cell death. Both enzymes likewise de-ubiquitinate TRAF2, a ubiquitin-ligase in the TNFR1 complex. Whilst this and the similar sub-cellular localisations of both enzyme isoforms indicate a substantial overlap of activities, inactivation by RNAi revealed that only the knock-down of USP2c resulted in apoptosis, whilst targeting USP2a did not have any consequence on the cells' survival. Consequently, we focussed our studies on USP2a and found that TRAF2 inhibits USP2a's effect on K48- but not on K63-linked ubiquitin chains. Hence, the ratio between USP2a and TRAF2 protein levels determines the cells' sensitivity to cell death.     

NODULE ROOT and COCHLEATA Maintain Nodule Development and Are Legume Orthologs of Arabidopsis BLADE-ON-PETIOLE Genes

During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.     

Etk/BMX, a Btk family tyrosine kinase, and Mal contribute to the cross-talk between MyD88 and FAK pathways

MyD88 and focal adhesion kinase (FAK) are key adaptors involved in signaling downstream of TLR2, TLR4, and integrin alpha5beta1, linking pathogen-associated molecule detection to the initiation of proinflammatory response. The MyD88 and integrin pathways are interlinked, but the mechanism of this cross-talk is not yet understood. In this study we addressed the involvement of Etk, which belongs to the Tec family of tyrosine kinases, in the cross-talk between the integrin/FAK and the MyD88 pathways in fibroblast-like synoviocytes (FLS) and in IL-6 synthesis. Using small interfering RNA blockade, we report that Etk plays a major role in LPS- and protein I/II (a model activator of FAK)-dependent IL-6 release by activated FLS. Etk is associated with MyD88, FAK, and Mal as shown by coimmunoprecipitation. Interestingly, knockdown of Mal appreciably inhibited IL-6 synthesis in response to LPS and protein I/II. Our results also indicate that LPS and protein I/II induced phosphorylation of Etk and Mal in rheumatoid arthritis FLS via a FAK-dependent pathway. In conclusion, our data provide support that, in FLS, Etk and Mal are implicated in the cross-talk between FAK and MyD88 and that their being brought into play is clearly dependent on FAK.     

Characterization of a bacteriocin-producing strain of Enterococcus faecalis from cow’s milk used in the production of Moroccan traditional dairy foods

A bacteriocin-producing lactic acid bacterium (strain 2.5) isolated from cow’s milk used in cheese production from Northern Morocco was selected for its strong anti-listerial activity. The producer strain was identified as Enterococcus faecalis by molecular methods. Strain 2.5 carried the genetic determinants for the two-peptide enterococcal bacteriocin enterocin 1071, and the active bacteriocin was purified to homogeneity by reversed-phase chromatography from culture broths of the producer strain. Strain 2.5 carried two plasmids (of ∼7 and 40 kb). Characterization of strain 2.5 at biosafety level indicated that this strain is non-haemolytic, and lacks the genetic determinants for most of the virulence factors described in enterococci (cylB, cylM, gelE, ace and agg) although it carried the genetic determinants cylA, efaAfs as well as determinants for the sex pheromone peptides cpd, cob, and ccf. Strain 2.5 was resistant to tetracycline, rifampicin, and ciprofloxacin, but it was sensitive to penicillin, ampicillin, vancomycin, and teicoplanin. Results from the present study support the potential role of strain 2.5 as an anti-listerial agent to be tested in traditional fermented foods.     

Design and evaluation of proniosomes as a carrier for ocular delivery of lomefloxacin HCl

The current investigation aims to develop and evaluate novel ocular proniosomal gels of lomefloxacin HCl (LXN); in order to improve its ocular bioavailability for the management of bacterial conjunctivitis. Proniosomes were prepared using different types of nonionic surfactants solely and as mixtures with Span 60. The formed gels were characterized for entrapment efficiency, vesicle size, and in vitro drug release. Only Span 60 was able to form stable LXN-proniosomal gel when used individually while the other surfactants formed gels only in combination with Span 60 at different ratios. The optimum proniosomal gel; P-LXN 7 (Span 60:Tween 60, 9:1) appeared as spherical shaped vesicles having high entrapment efficiency (>80%), appropriate vesicle size (187?nm) as well as controlled drug release over 12?h. Differential scanning calorimetry confirmed the amorphous nature of LXN within the vesicles. Stability study did not show any significant changes in entrapment efficiency or vesicle size after storage for 3 months at 4?°C. P-LXN 7 was found to be safe and suitable for ocular delivery as proven by the irritancy test. The antibacterial activity of P-LXN 7 evaluated using the susceptibility test and topical therapy of induced ocular conjunctivitis confirmed the enhanced antibacterial therapeutic efficacy of the LXN-proniosomal gel compared to the commercially available LXN eye drops.