Isolation and Chimerization of a Highly Neutralizing Antibody Conferring Passive Protection against Lethal Bacillus anthracis Infection

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD₅₀ B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD₅₀ of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.     

Identification and Localization of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae)

Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor “Candidatus Portiera aleyrodidarum,” an obligatory symbiotic bacterium which is housed in a special organ called the bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.     

Novel Rickettsiella Bacterium in the Leafhopper Orosius albicinctus (Hemiptera: Cicadellidae)

Bacteria in the genus Rickettsiella (Coxiellaceae), which are mainly known as arthropod pathogens, are emerging as excellent models to study transitions between mutualism and pathogenicity. The current report characterizes a novel Rickettsiella found in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae), a major vector of phytoplasma diseases in Europe and Asia. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to survey the main symbionts of O. albicinctus, revealing the obligate symbionts Sulcia and Nasuia, and the facultative symbionts Arsenophonus and Wolbachia, in addition to Rickettsiella. The leafhopper Rickettsiella is allied with bacteria found in ticks. Screening O. albicinctus from the field showed that Rickettsiella is highly prevalent, with over 60% of individuals infected. A stable Rickettsiella infection was maintained in a leafhopper laboratory colony for at least 10 generations, and fluorescence microscopy localized bacteria to accessory glands of the female reproductive tract, suggesting that the bacterium is vertically transmitted. Future studies will be needed to examine how Rickettsiella affects host fitess and its ability to vector phytopathogens.     

Stimulation,Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase. (a) Thermal stability of the Na,K-ATPase depends crucially on a specific interaction with 18:0/18:1 phosphatidylserine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine; SOPS) and cholesterol, which strongly amplifies stabilization. We show here that cholesterol associates with SOPS, FXYD1, and the α subunit between trans-membrane segments αTM8 and -10 to stabilize the protein. (b) Polyunsaturated neutral lipids stimulate Na,K-ATPase turnover by >60%. A screen of the lipid specificity showed that 18:0/20:4 and 18:0/22:6 phosphatidylethanolamine (PE) are the optimal phospholipids for this effect. (c) Saturated phosphatidylcholine and sphingomyelin, but not saturated phosphatidylserine or PE, inhibit Na,K-ATPase activity by 70–80%. This effect depends strongly on the presence of cholesterol. Analysis of the Na,K-ATPase activity and E₁-E₂ conformational transitions reveals the kinetic mechanisms of these effects. Both stimulatory and inhibitory lipids poise the conformational equilibrium toward E₂, but their detailed mechanisms of action are different. PE accelerates the rate of E₁ → E₂P but does not affect E₂(2K)ATP → E₁3NaATP, whereas sphingomyelin inhibits the rate of E₂(2K)ATP → E₁3NaATP, with very little effect on E₁ → E₂P. We discuss these lipid effects in relation to recent crystal structures of Na,K-ATPase and propose that there are three separate sites for the specific lipid interactions, with potential physiological roles to regulate activity and stability of the pump.     

Peroxisystem: Harnessing systems cell biology to study peroxisomes

In recent years, high‐throughput experimentation with quantitative analysis and modelling of cells, recently dubbed systems cell biology, has been harnessed to study the organisation and dynamics of simple biological systems. Here, we suggest that the peroxisome, a fascinating dynamic organelle, can be used as a good candidate for studying a complete biological system. We discuss several aspects of peroxisomes that can be studied using high‐throughput systematic approaches and be integrated into a predictive model. Such approaches can be used in the future to study and understand how a more complex biological system, like a cell and maybe even ultimately a whole organism, works.     

Study of 'Redhaven' peach and its white-fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism

Background  

Carotenoids are plant metabolites which are not only essential in photosynthesis but also important quality factors in determining the pigmentation and aroma of flowers and fruits. To investigate the regulation of carotenoid metabolism, as related to norisoprenoids and other volatile compounds in peach (Prunus persica L. Batsch.), and the role of carotenoid dioxygenases in determining differences in flesh color phenotype and volatile composition, the expression patterns of relevant carotenoid genes and metabolites were studied during fruit development along with volatile compound content. Two contrasted cultivars, the yellow-fleshed 'Redhaven' (RH) and its white-fleshed mutant 'Redhaven Bianca' (RHB) were examined.     

Resolving diurnal dynamics of the chloroplastic glutathione redox state in Arabidopsis reveals its photosynthetically derived oxidation

Plants are subjected to fluctuations in light intensity, and this might cause unbalanced photosynthetic electron fluxes and overproduction of reactive oxygen species (ROS). Electrons needed for ROS detoxification are drawn, at least partially, from the cellular glutathione (GSH) pool via the ascorbate–glutathione cycle. Here, we explore the dynamics of the chloroplastic glutathione redox potential (chl-E_GSH) using high-temporal-resolution monitoring of Arabidopsis (Arabidopsis thaliana) lines expressing the reduction–oxidation sensitive green fluorescent protein 2 (roGFP2) in chloroplasts. This was carried out over several days under dynamic environmental conditions and in correlation with PSII operating efficiency. Peaks in chl-E_GSH oxidation during dark-to-light and light-to-dark transitions were observed. Increasing light intensities triggered a binary oxidation response, with a threshold around the light saturating point, suggesting two regulated oxidative states of the chl-E_GSH. These patterns were not affected in npq1 plants, which are impaired in non-photochemical quenching. Oscillations between the two oxidation states were observed under fluctuating light in WT and npq1 plants, but not in pgr5 plants, suggesting a role for PSI photoinhibition in regulating the chl-E_GSH dynamics. Remarkably, pgr5 plants showed an increase in chl-E_GSH oxidation during the nights following light stresses, linking daytime photoinhibition and nighttime GSH metabolism. This work provides a systematic view of the dynamics of the in vivo chloroplastic glutathione redox state during varying light conditions.

Monitoring the daily in vivo dynamics of the chloroplastic GSH redox state in light-stressed wild-type plants versus photoprotective mutants provides insight into the photosynthesis-dependent production of oxidants.     

Are selective serotonin reuptake inhibitors cardiac teratogens? Echocardiographic screening of newborns with persistent heart murmur

BACKGROUND : Selective serotonin reuptake inhibitors (SSRIs) have been suspected of cardiac teratogenicity, but reports have been inconsistent. Our aim was to investigate the rate of nonsyndromic congenital heart defects in newborns exposed in utero to SSRIs compared with unexposed controls. METHODS : This prospective study of women who gave birth at our tertiary center from 2000 to 2007 yielded 235 women who reported first‐trimester SSRI use during pregnancy. All newborns born during the study period and found to have a persistent cardiac murmur on day 2 or 3 of life were referred for examination by a pediatric cardiologist and by echocardiography. The findings were compared between the newborns who were exposed to SSRIs and those who were not. RESULTS : Nonsyndromic congenital heart defects were identified by echocardiography in 8 of 235 (3.40%) newborns exposed in utero to SSRIs and in 1083 of 67,636 (1.60%) non‐exposed newborns. The difference in prevalence between the two groups was significant (relative risk, 2.17; 95% confidence interval, 1.07–4.39). The prevalence rates for paroxetine and fluoxetine exposure were 4.3% and 3.0%, respectively. All cardiac defects in the study group were mild: ventricular septal defect (6), bicuspid aortic valve (1) and right superior vena cava to coronary sinus (1). CONCLUSIONS : Newborns exposed in utero to SSRIs, have a twofold higher risk of mild nonsyndromic heart defects than unexposed infants. The data suggest that women who require SSRI treatment during pregnancy can be reassured that the fetal risk is low and possible cardiac malformations will probably be mild. Late‐targeted ultrasound and fetal echocardiography at 22 to 23 weeks' gestation are recommended in this patient group. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

The availability of filament ends modulates actin stochastic dynamics in live plant cells

A network of individual filaments that undergoes incessant remodeling through a process known as stochastic dynamics comprises the cortical actin cytoskeleton in plant epidermal cells. From images at high spatial and temporal resolution, it has been inferred that the regulation of filament barbed ends plays a central role in choreographing actin organization and turnover. How this occurs at a molecular level, whether different populations of ends exist in the array, and how individual filament behavior correlates with the overall architecture of the array are unknown. Here we develop an experimental system to modulate the levels of heterodimeric capping protein (CP) and examine the consequences for actin dynamics, architecture, and cell expansion. Significantly, we find that all phenotypes are the opposite for CP-overexpression (OX) cells compared with a previously characterized cp-knockdown line. Specifically, CP OX lines have fewer filament–filament annealing events, as well as reduced filament lengths and lifetimes. Further, cp-knockdown and OX lines demonstrate the existence of a subpopulation of filament ends sensitive to CP concentration. Finally, CP levels correlate with the biological process of axial cell expansion; for example, epidermal cells from hypocotyls with reduced CP are longer than wild-type cells, whereas CP OX lines have shorter cells. On the basis of these and other genetic studies in this model system, we hypothesize that filament length and lifetime positively correlate with the extent of axial cell expansion in dark-grown hypocotyls.