Effect of the charge distribution along the “ferritin-like” pores of the proteins from the Dps family on the iron incorporation process

DNA-binding proteins from starved cells (Dps) differ in the number and position of charged residues along the “ferritin-like” pores that are used by iron to reach the ferroxidase center and the protein cavity. These differences are shown to affect significantly the electrostatic potential at the pores, which determines the extent of cooperativity in the iron uptake kinetics and thereby the mass distribution of the ferric hydroxide micelles inside the protein cavity. These conclusions are of biotechnological value in the preparation of protein-enclosed nanomaterials and are expected to apply also to ferritins. They were reached after characterization of the Dps from Listeria innocua, Helicobacter pylori, Thermosynechococcus elongatus, Escherichia coli, and Mycobacterium smegmatis. The characterization comprised the calculation of the electrostatic potential at the pores, determination of the iron uptake kinetics in the presence of molecular oxygen or hydrogen peroxide, and analysis of the proteins by means of the sedimentation velocity after iron incorporation.     

Lipopolysaccharide- and lipoteichoic acid-induced tolerance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase

Human Toll-like receptor (TLR) 4 and TLR2 receptors recognize LPS or lipoteichoic acid (LTA), respectively. Prolonged exposure of human macrophages/monocytes to bacterial LPS induces a state of adaptation/tolerance to subsequent LPS challenge. Inflammatory gene expressions such as IL-1beta and TNF-alpha are selectively repressed, while certain anti-inflammatory genes such as secretory IL-1R antagonist are still induced in LPS-adapted/tolerant cells. In this report, we demonstrate that LPS-tolerized human promonocytic THP-1 cells develop cross-tolerance and no longer respond to LTA-induced IL-1beta/TNF-alpha production, indicating that disruption of common intracellular signaling is responsible for the decreased IL-1beta/TNF-alpha production. We observe that down-regulation of IL-1R-associated kinase (IRAK) protein level and kinase activity closely correlates with the development of cross-tolerance. IRAK protein levels and kinase activities in LPS-tolerized cells remain low and hyporesponsive to subsequent LPS or LTA challenges. We also demonstrate that THP-1 cells with prolonged LTA treatment develop LTA tolerance and do not express IL-1beta/TNF-alpha upon further LTA challenge. Strikingly, cells tolerized with LTA are only refractory to subsequent LTA challenge and can still respond to LPS stimulation. Correspondingly, stimulation of TLR2 by LTA, although activating IRAK, does not cause IRAK degradation. IRAK from LTA-tolerized cells can be subsequently activated and degraded by further LPS challenge, but not LTA treatment. Our studies reveal that LTA-induced tolerance is distinct compared with that of LPS tolerance, and is likely due to disruption of unique TLR2 signaling components upstream of MyD88/IRAK.     

Fed-batch process for the psychrotolerant marine bacterium <Emphasis Type=Italic>Pseudoalteromonas haloplanktis</Emphasis>

Background  

Pseudoalteromonas haloplanktis is a cold-adapted γ-proteobacterium isolated from Antarctic sea ice. It is characterized by remarkably high growth rates at low temperatures. P. haloplanktis is one of the model organisms of cold-adapted bacteria and has been suggested as an alternative host for the soluble overproduction of heterologous proteins which tend to form inclusion bodies in established expression hosts. Despite the progress in establishing P. haloplanktis as an alternative expression host the cell densities obtained with this organism, which is unable to use glucose as a carbon source, are still low. Here we present the first fed-batch cultivation strategy for this auspicious alternative expression host.     

The long-distance abscisic acid signal in the droughted plant: the fate of the hormone on its way from root to shoot

Abscisic acid (ABA) is a potent molecule that certainly modifies stomatal behaviour and plant water loss and probably acts to modify the growth of leaves. The hormone is synthesized both in the leaves and the roots of the plant and in the soil and may move freely from plant to soil and soil to plant. It can also move rapidly through the plant in both the xylem and the phloem and will partition between different compartments in different tissues largely as a function of pH. It is described here how perturbations in soil conditions around the roots and the water status of the air can modify the fluxes of ABA around the plant and its accumulation in different compartments and different tissues. These fluxes can be interpreted as signals of different stresses imposed on the plant and consideration is given to how different perturbations can exert subtle changes which are manifest as modified shoot growth rates and functioning. Most emphasis in the discussion is placed upon the plant's responses to the imposition of soil and atmospheric drought.     

Expression of Green Fluorescent Protein in <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> Is Affected by Passage Through Host Plants

Xylella fastidiosa, a Gram-negative bacterial plant pathogen, causes Pierces disease of grapevine in North America. In South America the pathogen causes citrus variegated chlorosis, which is widespread in Brazil. We have introduced into Xylella fastidiosa a mini-Tn5 transposon that encodes a green fluorescent protein (GFP) gene optimized for expression in bacteria. The mini-Tn5 derivative was inserted into different sites of the genome in independent transconjugants as determined by Southern blotting. The GFP gene was expressed well and to different levels in different transconjugants. Four independent transconjugants were separately used to inoculate sweet orange and tobacco seedlings. The transconjugants were able to colonize the plants and were subsequently isolated from points distal to the inoculation sites. When the relative fluorescence of the transconjugants that had been passed through either tobacco or sweet orange was compared with that of the same transconjugant maintained continuously in vitro, we observed that passage through either plant host significantly increased the level of expression of the GFP. The increased level of expression of GFP was transient, and was lost upon further culture in vitro. Xylella fastidiosa forms biofilms in planta which are believed to represent a metabolically differentiated state. The increased expression of GFP observed after passage through plants may be accounted for by this phenomenon.     

Contribution of store-operated Ca2+ entry to pHo-dependent changes in vascular tone of porcine coronary smooth muscle

Vascular smooth muscle contracts on increases of extracellular pH (pH(o)) and relaxes on pH(o) decreases possibly resulting from changes in transsarcolemmal Ca(2+) influx. Therefore, we studied store-operated Ca(2+) entry (SOCE; i.e. capacitative Ca(2+) entry (CCE)) during acidification (pH(o)=6.5) and alkalinization (pH(o)=8.0) in isolated porcine coronary smooth muscle cells (SMCs) by monitoring cytoplasmic Ca(2+) ([Ca(2+)](i)) and divalent cation entry (Mn(2+) quench) with fura-2/AM-fluorometry. Additionally, we evaluated the contribution of SOCE to pH(o)-dependent changes in isometric tension of porcine coronary smooth muscle strips. SOCE elicited in SMCs by the SERCA inhibitor BHQ was strongly modulated by pH(o) showing a decrease upon acidification and vice versa an increase upon alkalinization. BHQ-mediated tension of smooth muscle strips also revealed strong pH(o) dependence. In contrast, L-VOC-dependent tension ([K(+)](o)=20 and 40 mmol l(-1)) was remarkably less affected by pH(o) changes. Moreover, refilling of depleted Ca(2+) stores after repeated M(3)-cholinergic receptor stimulation could be almost completely inhibited by SKF 96365 and was markedly reduced by acidification and considerably enhanced by alkalinization pointing to a major role of SOCE in refilling. We conclude that vascular tone particularly responds to alterations in pH(o) whenever SOCE substantially contributes to the amount of activator Ca(2+) for contraction.     

Water flows in the parasitic association Rhinanthus minor/Hordeum vulgare

Using the facultative root hemiparasite Rhinanthus minor and its host Hordeum vulgare several aspects of water relations have been measured in this parasitic association. Extraction of xylem sap by the parasite from the host's roots is facilitated by con siderably higher transpiration per leaf area in the parasite than in the host and by the fact that stomata of attached Rhinanthus were open all day and night despite extremely high ABA concentrations in the leaves. By comparison, another root hemiparasite, Melampyrum arvense, parasitizing various grasses in the field, showed normal diurnal stomatal behaviour. The abnormal behaviour of Rhinanthus stomata was not due to anatomical reasons as closure could be induced by applying high external ABA concentrations. Remarkable differences have been detected between the hydraulic conductance of barley seminal roots showing relatively low values and that of Rhinanthus seminal roots showing very high values. The latter could be related to the observed high ABA concentrations in these roots. Whole plant water uptake, transpirational losses, growth-dependent deposition, and the flows of water within the plants have been measured in singly growing Rhinanthus and Hordeum plants and in the parasitic association between the two. Water uptake, deposition and transpiration in Rhinanthus were dramatically increased after attachment to the barley host; most of the water used by the parasite was extracted as xylem sap from the host, thereby scavenging 20% of the total water taken up by the host's roots. This water uptake by the parasitized host, however, due to a parasite-induced reduction in the host's growth, was decreased by 22% as compared to non-parasitized barley. The overall changes in growth-related water deposition in the host and parasite pointed to decreased shoot growth and relatively favoured root growth in the host and to strongly favoured shoot growth in the parasite. These changes in the host became more severe, when more than one Rhinanthus was parasitizing one barley plant.     

Profound differences in leukocyte-endothelial cell responses to lipopolysaccharide versus lipoteichoic acid

We have investigated the effects of LPS from Escherichia coli, lipoteichoic acid (LTA), and peptidoglycan (PepG) from Staphylococcus aureus, and live S. aureus on leukocyte-endothelial interactions in vivo using intravital microscopy to visualize muscle microvasculature. Systemic vs local administration of LPS induced very different responses. Local administration of LPS into muscle induced significant leukocyte rolling, adhesion, and emigration in postcapillary venules at the site of injection. LPS given systemically dramatically dropped circulating leukocyte counts and increased neutrophils in the lung. However, the drop in circulating leukocytes was not associated with leukocyte sequestration to the site of injection (peritoneum) nor to peripheral microvessels in muscles. Unlike LPS, various preparations of LTA had no systemic and very minor local effect on leukocyte-endothelial interactions, even at high doses and for prolonged duration. LPS, but not LTA, potently activated human endothelium to recruit leukocytes under flow conditions in vitro. Endothelial adhesion molecule expression was also increased extensively with LPS, but not LTA. Interestingly, systemic administration of live S. aureus induced leukocyte-endothelial cell responses similar to LPS. PepG was able to induce leukocyte-endothelial interactions in muscle and peritoneum, but had no effect systemically (no increase in neutrophils in lungs and no decrease in circulating neutrophil counts). These results demonstrate that: 1) LPS has potent, but divergent local and systemic effects on leukocyte-endothelial interactions; 2) S. aureus can induce a systemic response similar to LPS, but this response is unlikely to be due to LTA, but more likely to be mediated in part by PepG.