Protein phosphorylation in the facultative chemolithotrophThiobacillus novellus

The phosphorylation of at least five proteins with M_r of about 160,000; 93,000; 85,000; 45,000; and 29,000 respectively was demonstrated in crude extracts from the facultative chemolithotrophThiobacillus novellus. The incorporation of [-³²P]phosphate from ATP into these proteins was dependent on the presence of magnesium ion. The phosphorylation reactions were found to be reversible and required 12.5 mM NaF for maximal activity, indicating the action of phosphatases. In addition, 3,5-cAMP had little effect on protein kinase activity, whereas Ca²⁺ alone was weakly stimulatory. This activation was enhanced by the addition of 3,5-cAMP. Ca²⁺ with calmodulin had a strong stimulatory effect on phosphate incorporation into the proteins. A highly purified preparation containing only the 160, 93, and 85 kDa proteins phosphorylated histone, whereas the uptake of³²P by the three proteins was inhibited. Rabbit muscle phosphorylase b prevented incorporation of radiolabel only into the 160 and 93 kDa proteins.     

A Phase 1b Randomized,Controlled, Double-Blinded Dosage-Escalation Trial to Evaluate the Safety,Reactogenicity and Immunogenicity of an Adenovirus Type 35 Based Circumsporozoite Malaria Vaccine in Burkinabe Healthy Adults 18 to 45 Years of Age

Background

Ad35.CS.01 is a pre-erythrocytic malaria candidate vaccine. It is a codon optimized nucleotide sequence representing the P. falciparum circumsporozoite (CS) surface antigen inserted in a replication deficient Adenovirus 35 backbone. A Phase 1a trial has been conducted in the USA in naïve adults and showed that the vaccine was safe. The aim of this study is to assess the safety and immunogenicity of ascending dosages in sub Saharan Africa.

Methods

A double blind, randomized, controlled, dose escalation, phase Ib trial was conducted in a rural area of Balonghin, the Saponé health district (Burkina Faso). Forty-eight healthy adults aged 18-45 years were randomized into 4 cohorts of 12 to receive three vaccine doses (day 0, 28 and 84) of 10⁹, 10¹⁰, 5X10¹⁰, 10¹¹ vp of Ad35.CS.01 or normal saline by intra muscular injection. Subjects were monitored carefully during the 14 days following each vaccination for non serious adverse events. Severe and serious adverse events were collected throughout the participant study duration (12 months from the first vaccination). Humoral and cellular immune responses were measured on study days 0, 28, 56, 84, 112 and 140.

Results

Of the forty-eight subjects enrolled, forty-four (91.7%) received all three scheduled vaccine doses. Local reactions, all of mild severity, occurred in thirteen (27.1%) subjects. Severe (grade 3) laboratory abnormalities occurred in five (10.4%) subjects. One serious adverse event was reported and attributed to infection judged unrelated to vaccine. The vaccine induced both antibody titers and CD8 T cells producing IFNγ and TNFα with specificity to CS while eliciting modest neutralizing antibody responses against Ad35.

Conclusion

Study vaccine Ad35.CS.01 at four different dose levels was well-tolerated and modestly immunogenic in this population. These results suggest that Ad35.CS.01 should be further investigated for preliminary efficacy in human challenge models and as part of heterologous prime-boost vaccination strategies.

Trial Registration

ClinicalTrials.gov NCT01018459 http://clinicaltrials.gov/ct2/show/NCT01018459     

Hormonal Therapy and Risk of Breast Cancer in Mexican Women

The use of hormonal therapies, including hormonal contraceptives (HC) and postmenopausal hormone replacement therapy (HRT) have been shown to influence breast cancer (BC) risk. However, the variations of these effects among populations and ethnic groups are not completely documented, especially among Hispanic women. We evaluated the association between HC and premenopausal BC risk, and between HRT and postmenopausal BC risk in Mexican women. Data from a Mexican multi-center population-based case–control study ofwomen aged 35 to 69 years were analysed. A total of 1000 cases and 1074 matched controls were recruited between 2004 and 2007. Information on hormonal therapy was collected through a structured questionnaire. Results were analysed using conditional logistic regression models. Overall, HC were used by 422/891 (47.3%) premenopausal women and HRT was used by 220/1117 (19.7%) postmenopausal women. For HC, odds ratios (ORs) for BC were 1.11 (95% confidence interval (CI): 0.82, 1.49) for current users and 1.68 (95% CI: 0.67, 4.21) for ever-users. No clear effect of duration of use was observed. For HRT, the OR for BC was significantly increased in ever users (OR: 1.45; 95% CI: 1.01, 2.08). A non-significant increased risk was observed for combined estrogen/progestin, (OR =  1.85; 95% CI: 0.84, 4.07) whereas no effect was observed for the use of estrogen alone (OR = 1.14; 95% CI: 0.68, 1.91). Our results indicate that, HC had a non-significant effect on the risk of pre-menopausal BC, but suggested that injected contraceptives may slightly increase the risk, whereas HRT had a significant effect on post-menopausal BC in this population. This study provides new information about the effects of HC and HRT on BC risk in a Mexican population, which may be of relevance for the population of Latin America as a whole.     

Dynamic microtubules produce an asymmetric E-cadherin–Bazooka complex to maintain segment boundaries

Distributing junctional components around the cell periphery is key for epithelial tissue morphogenesis and homeostasis. We discovered that positioning of dynamic microtubules controls the asymmetric accumulation of E-cadherin. Microtubules are oriented preferentially along the dorso-ventral axis in Drosophila melanogaster embryonic epidermal cells, and thus more frequently contact E-cadherin at dorso-ventral cell–cell borders. This inhibits RhoGEF2, reducing membrane recruitment of Rho-kinase, and increasing a specific E-cadherin pool that is mobile when assayed by fluorescence recovery after photobleaching. This mobile E-cadherin is complexed with Bazooka/Par-3, which in turn is required for normal levels of mobile E-cadherin. Mobile E-cadherin–Bazooka prevents formation of multicellular rosette structures and cell motility across the segment border in Drosophila embryos. Altogether, the combined action of dynamic microtubules and Rho signaling determines the level and asymmetric distribution of a mobile E-cadherin–Bazooka complex, which regulates cell behavior during the generation of a patterned epithelium.     

Protection against cardiac injury by small Ca2 +-sensitive K+ channels identified in guinea pig cardiac inner mitochondrial membrane

We tested if small conductance, Ca^2 +‐sensitive K⁺ channels (SK_Ca) precondition hearts against ischemia reperfusion (IR) injury by improving mitochondrial (m) bioenergetics, if O₂‐derived free radicals are required to initiate protection via SK_Ca channels, and, importantly, if SK_Ca channels are present in cardiac cell inner mitochondrial membrane (IMM). NADH and FAD, superoxide (O₂^?), and m[Ca^2 +] were measured in guinea pig isolated hearts by fluorescence spectrophotometry. SK_Ca and IK_Ca channel opener DCEBIO (DCEB) was given for 10 min and ended 20 min before IR. Either TBAP, a dismutator of O₂^?, NS8593, an antagonist of SK_Ca isoforms, or other K_Ca and K_ATP channel antagonists, were given before DCEB and before ischemia. DCEB treatment resulted in a 2-fold increase in LV pressure on reperfusion and a 2.5 fold decrease in infarct size vs. non-treated hearts associated with reduced O₂^? and m[Ca^2 +], and more normalized NADH and FAD during IR. Only NS8593 and TBAP antagonized protection by DCEB. Localization of SK_Ca channels to mitochondria and IMM was evidenced by a) identification of purified mSK_Ca protein by Western blotting, immuno-histochemical staining, confocal microscopy, and immuno-gold electron microscopy, b) 2-D gel electrophoresis and mass spectroscopy of IMM protein, c) [Ca^2 +]‐dependence of mSK_Ca channels in planar lipid bilayers, and d) matrix K⁺ influx induced by DCEB and blocked by SK_Ca antagonist UCL1684. This study shows that 1) SK_Ca channels are located and functional in IMM, 2) mSK_Ca channel opening by DCEB leads to protection that is O₂^? dependent, and 3) protection by DCEB is evident beginning during ischemia.     

Coronin 1B supports RhoA signaling at cell-cell junctions through Myosin II

Non-muscle myosin II (NMII) motor proteins are responsible for generating contractile forces inside eukaryotic cells. There is also a growing interest in the capacity for these motor proteins to influence cell signaling through scaffolding, especially in the context of RhoA GTPase signaling. We previously showed that NMIIA accumulation and stability within specific regions of the cell cortex, such as the zonula adherens (ZA), allows the formation of a stable RhoA signaling zone. Now we demonstrate a key role for Coronin 1B in maintaining this junctional pool of NMIIA, as depletion of Coronin 1B significantly compromised myosin accumulation and stability at junctions. The loss of junctional NMIIA, upon Coronin 1B knockdown, perturbed RhoA signaling due to enhanced junctional recruitment of the RhoA antagonist, p190B Rho GAP. This effect was blocked by the expression of phosphomimetic MRLC-DD, thus reinforcing the central role of NMII in regulating RhoA signaling.     

Eimeria tenella ROP kinase EtROP1 induces G0/G1 cell cycle arrest and inhibits host cell apoptosis

Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the aetiologic agent of caecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterised. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N‐terminal extension is necessary for its catalytic kinase activity. Ectopic expression of EtROP1 followed by co‐immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterisation confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest. This work functionally described the first ROP kinase from E. tenella and its noncanonical structure. Our study provides the first mechanistic insight into host cell apoptosis inhibition by E. tenella. EtROP1 appears as a new candidate for coccidiosis control.