Plant cell nucleolus as a hot spot for iron

Many central metabolic processes require iron as a cofactor and take place in specific subcellular compartments such as the mitochondrion or the chloroplast. Proper iron allocation in the different organelles is thus critical to maintain cell function and integrity. To study the dynamics of iron distribution in plant cells, we have sought to identify the different intracellular iron pools by combining three complementary imaging approaches, histochemistry, micro particle-induced x-ray emission, and synchrotron radiation micro X-ray fluorescence. Pea (Pisum sativum) embryo was used as a model in this study because of its large cell size and high iron content. Histochemical staining with ferrocyanide and diaminobenzidine (Perls/diaminobenzidine) strongly labeled a unique structure in each cell, which co-labeled with the DNA fluorescent stain DAPI, thus corresponding to the nucleus. The unexpected presence of iron in the nucleus was confirmed by elemental imaging using micro particle-induced x-ray emission. X-ray fluorescence on cryo-sectioned embryos further established that, quantitatively, the iron concentration found in the nucleus was higher than in the expected iron-rich organelles such as plastids or vacuoles. Moreover, within the nucleus, iron was particularly accumulated in a subcompartment that was identified as the nucleolus as it was shown to transiently disassemble during cell division. Taken together, our data uncover an as yet unidentified although abundant iron pool in the cell, which is located in the nuclei of healthy, actively dividing plant tissues. This result paves the way for the discovery of a novel cellular function for iron related to nucleus/nucleolus-associated processes.     

One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification

We describe a novel cloning method, referred to as insert-tagged (InTag) positive selection, for the rapid one-step reformatting of phage-displayed antibody fragments to full-length immunoglobulin Gs (IgGs). InTag positive selection enables recombinant clones of interest to be directly selected without cloning background, bypassing the laborious process of plating out cultures and colony screening and enabling the cloning procedure to be automated and performed in a high-throughput format. This removes a significant bottleneck in the functional screening of phage-derived antibody candidates and enables a large number of clones to be directly reformatted into IgG without the intermediate step of Escherichia coli expression and testing of soluble antibody fragments. The use of InTag positive selection with the Dyax Fab-on-phage antibody library is demonstrated, and optimized methods for the small-scale transient expression of IgGs at high levels are described. InTag positive selection cloning has the potential for wide application in high-throughput DNA cloning involving multiple inserts, markedly improving the speed and quality of selections from protein libraries.     

The Coagulation Factor XIIa Inhibitor rHA-Infestin-4 Improves Outcome after Cerebral Ischemia/Reperfusion Injury in Rats

Background and Purpose

Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach.

Methods

For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation.

Results

Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis.

Conclusions

With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.     

An analysis of class I antigens of man and other species by one-dimensional IEF and immunoblotting

A procedure for the molecular identification of MHC class I products based on 1-D IEF and subsequent immunoblotting is described. Optimal conditions for 1-D IEF, the electrophoretic transfer of proteins out of denaturing, nonionic detergent-containing gels to nitrocellulose, and the requisite antibodies, both polyclonal and monoclonal, for the visualization of class I heavy chains have been established. Cross-reactivity of antibodies has enabled the biochemical analysis of class I heavy chains in the dog. The procedure reported here requires modest amounts of cells and allows a rapid molecular characterization of class I heavy chain polymorphisms in man and other species without the need for radiochemical methods.Abbreviations used in this paper FCS fetal calf serum - MHC major histocompatibility complex - NP-40 Nonidet P-40 - PBL peripheral blood lymphocytes - PHA phytohemagglutinin - RaHC rabbit anti-heavy chain serum - TX-114 Triton X-114 - 1-D IEF one-dimensional isoelectric focusing     

Aromatic residues in the C-terminal helix of human apoC-I mediate phospholipid interactions and particle morphology

Human apolipoprotein C-I (apoC-I) is an exchangeable apolipoprotein that binds to lipoprotein particles in vivo. In this study, we employed a LC-MS/MS assay to demonstrate that residues 38-51 of apoC-I are significantly protected from proteolysis in the presence of 1,2-dimyristoyl-3-sn-glycero-phosphocholine (DMPC). This suggests that the key lipid-binding determinants of apoC-I are located in the C-terminal region, which includes F42 and F46. To test this, we generated site-directed mutants substituting F42 and F46 for glycine or alanine. In contrast to wild-type apoC-I (WT), which binds DMPC vesicles with an apparent Kd [Kd(app)] of 0.89 microM, apoC-I(F42A) and apoC-I(F46A) possess 2-fold weaker affinities for DMPC with Kd(app) of 1.52 microM and 1.58 microM, respectively. However, apoC-I(F46G), apoC-I(F42A/F46A), apoC-I(F42G), and apoC-I(F42G/F46G) bind significantly weaker to DMPC with Kd(app) of 2.24 microM, 3.07 microM, 4.24 microM, and 10.1 microM, respectively. Sedimentation velocity studies subsequently show that the protein/DMPC complexes formed by these apoC-I mutants sediment at 6.5S, 6.7S, 6.5S, and 8.0S, respectively. This is compared with 5.0S for WT apoC-I, suggesting the shape of the particles was different. Transmission electron microscopy confirmed this assertion, demonstrating that WT forms discoidal complexes with a length-to-width ratio of 2.57, compared with 1.92, 2.01, 2.16, and 1.75 for apoC-I(F42G), apoC-I(F46G), apoC-I(F42A/F46A), and apoC-I(F42G/F46G), respectively. Our study demonstrates that the C-terminal amphipathic alpha-helix of human apoC-I contains the major lipid-binding determinants, including important aromatic residues F42 and F46, which we show play a critical role in stabilizing the structure of apoC-I, mediating phospholipid interactions, and promoting discoidal particle morphology.     

The mechanical binding strengths of Helicobacter pylori BabA and SabA adhesins using an adhesion binding assay–ELISA,and its clinical relevance in Japan

To elucidate a potential role for H. pylori BabA and SabA adhesins in the pathogenesis of gastric mucosal lesions, the MBS of BabA and SabA was examined using an in‐house ABA‐ELISA. Ninety isolates from Japanese patients with gastric cancer (n= 43) and non‐cancerous (n= 47) lesions were subjected to an ABA‐ELISA which had been developed in‐house, and sequential analysis of the babA2 middle region. The BabA‐MBS was significantly higher in the cancer than the non‐cancer group (P= 0.019), but there was no significant difference for SabA‐MBS. A weak correlation between BabA‐MBS and SabA‐MBS (r= 0.418) was observed, the positive correlation being higher in the cancer than the non‐cancer group (r= 0.598 and 0.288, respectively). The isolates were classified into two groups: a BabA‐high‐binding and a BabA‐low‐binding group (in comparison to the average for BabA‐MBS). The average SabA‐MBS in the BabA‐high‐binding group was significantly higher than in the BabA‐low‐binding group (P < 0.0001). Analysis of babA2 middle region diversity (AD1–5) revealed that AD2‐type was predominant in isolates irrespective of BabA‐MBS. H. pylori BabA‐MBS might have an effect on SabA‐MBS and relate to the severity of gastric disorders, including gastric cancer. Evaluation of MBS of the combined two adhesins would be helpful for predicting damage in the H. pylori infected stomach.     

Straightforward histochemical staining of Fe by the adaptation of an old-school technique: Identification of the endodermal vacuole as the site of Fe storage in Arabidopsis embryos

Iron (Fe) is an essential metal ion, required for basic cellular processes such as respiration, photosynthesis and cell division. Therefore, Fe has to be stored and distributed to several organelles to fulfill its roles. The molecular basis of Fe distribution is poorly understood. In this context, elemental imaging approaches are becoming essential for a better understanding of metal homeostasis in plants. Recently, several genes have been involved in Fe storage (VIT1) and remobilization (NRAMP3 and NRAMP4) in the seed of Arabidopsis, mostly with the help of sophisticated imaging techniques. We have adapted an histochemical procedure to detect Fe in plant tissues, based on Perls staining coupled to diaminobenzidine (DAB) intensification. The Perls/DAB technique, quick and inexpensive, was shown to be specific for Fe and highly sensitive. We have applied this procedure to Arabidopsis embryos and shown that Fe is stored in the vacuoles of a specific cell layer surrounding the pro-vascular system, the endodermis. Our results have revealed a new role for the endodermis in Fe storage in the embryo and established the Perls/DAB technique as a powerful tool to detect Fe in plant tissues and cells.Key words: iron, vacuole, Arabidopsis, endodermis, embryoIron (Fe) is a very important essential metal for all living organisms. The function of Fe in biological processes relies on its ability to exist in two redox states (ferrous and ferric iron). Consequently, Fe is crucial for metabolic reactions of respiration and photosynthesis. Although at the whole plant level the molecular mechanisms of Fe acquisition and storage are now well documented, the control of Fe distribution at organ, cell and sub-cellular levels is extremely poorly understood and represents an important stake. Recent discoveries on Fe storage and remobilization in the embryo have unequivocally shown that genetic approaches are not sufficient to unravel the function of genes, unless coupled to high-resolution metal imaging. The use of energy dispersive X-ray spectroscopy (EDX) and inelastically scattered electrons (ESI) on electron micrographs clearly showed that in Arabidopsis seeds Fe is accumulated in vacuoles and remobilized during germination.¹ Likewise, the analysis of Arabidopsis seeds by XRF and tomography has provided three-dimensional mapping of Fe that has showed that VIT1 (for Vacuolar Iron Transporter) is required for proper allocation of Fe in the vascular tissue of the embryo, a distribution that is altered in a vit1-1 mutant.² Noteworthy is the fact that these alterations of Fe distribution do not induce a change in total Fe content and thus would not have been detected by measurements of total Fe concentration. There is therefore an increasing interest for imaging techniques that are becoming a must-have to understand the function of metal transporters in plants. Metal imaging technologies are mostly based on X ray absorption/emission/fluorescence, often requiring expensive and rare equipments (synchrotron for instance). Taking advantage on the high reactivity of metal ions for organic ligands, several reagents have been used as chromophores for histochemical staining of metals. Among those, potassium ferrocyanide, also known as Perls reagent, was used since the late XIX^th century to produce the Prussian blue, after reaction with ferric iron. The Perls reagent has been widely used to stain Fe in tissues, but only occasionally in plants, due to its low sensitivity and poor penetration in hydrophobic tissues. Nevertheless, it is possible to increase the sensitivity of the staining by secondary reactions with diaminobenzidine (DAB) and hydrogen peroxide (H₂O₂). Indeed, since the Fe-Perls complex is redox active, the addition of DAB and H₂O₂ triggers the oxidative polymerization of DAB, producing brown pigments.³ This reaction is the basis of the intensification of the Perls staining (Perls/DAB from now on). To adapt this procedure to plants, we have first established that the staining was specific for Fe and did not practically cross-react with other metal ions. In doing so, we also showed that Perls/DAB could stain both Fe^II and Fe^III. We have chosen Arabidopsis seeds as model, in order to compare our Fe staining procedure with the imaging already available by XRF.² Compared to Perls stain alone, the Perls/DAB protocol appeared to be much more sensitive. Iron appeared to be concentrated around the provascular system of mature Arabidopsis embryos.Staining of the vit1-1 mutant showed a modified pattern compatible with the available μXRF-tomography data, thus clearly establishing that the staining procedure is specific for Fe in vivo and represents a new, quick and simple tool to detect Fe in plant samples.⁴ Thin sections of Perls/DAB embryos uncovered that, in mature embryos, Fe was concentrated in a single cell layer, apparently corresponding to the endodermis. This observation was further confirmed by the analysis of longitudinal sections of the radicle-hypocotyl region. In this particular zone, a second periclinal division occurs, giving rise to a cortex cell layer and the endodermis,⁵ the latter one alone being intensively stained with Perls/DAB. Other developmental mutants were used, such as for example the vein patterning mutant SCARFACE (scf),⁶ which presents Fe staining in cotyledons as small segments corresponding to discontinuous veins characteristic of the scf mutant (Fig. 1). Thus, the Perls/DAB protocol represents a very useful tool not only to study Fe homeostasis but also in the field of developmental research, as a marker of endodermis and provascular system of the embryo. Finally, the Perls/DAB procedure can be greatly improved by staining directly the histological thin sections instead of whole embryos, thereby (i) increasing tremendously the resolution and (ii) solving the problem of low penetration of the dyes in hydrophobic plant samples. This modification enabled us to show that in endodermal cells Fe is actually located in vacuoles. Remobilization of the vacuolar pool of iron by AtNRAMP3 and AtNRAMP4 is crucial during germination.¹ Since we found that Fe is blocked in the endodermis of the nramp3nramp4 mutant,⁴ we can now propose that in mature embryos Fe is mainly stored in the vacuoles of the endodermis.Open in a separate windowFigure 1Iron distribution in cotyledons. Wild-type and scf1 dry seed embryos were dissected and stained with Perls/DA B according to Roschzttardtz et al.⁴In conclusion, we have adapted an histochemical staining procedure, easy to set up and inexpensive, that fills the gap between the Perls reagent and X ray-based elemental imaging. The resolution, at the sub-cellular scale, makes it a valuable tool to investigate the distribution of Fe in plant tissues without employing electron microscopy or synchrotron X ray fluorescence. Furthermore, the possibility of direct staining on histological sections makes this technique applicable to virtually any plant material, after fixation and embedding in resin.     

Putative interhelical interactions within the PheP protein revealed by second-site suppressor analysis

Highly conserved glycine residues within span I and span II of the phenylalanine and tyrosine transporter PheP were shown to be important for the function of the wild-type protein. Replacement by amino acids with increasing side chain volume led to progressive loss of transport activity. Second-site suppression studies performed with a number of the primary mutants revealed a tight packing arrangement between spans I and II that is important for function and an additional interaction between spans I and III. We also postulate that a third motif, GXXIG, present in span I and highly conserved within different members of the amino acid-polyamine-organocation family, may function as a dimerization motif. Surprisingly, other highly conserved residues, such as Y60 and L41, could be replaced by various residues with no apparent loss of activity.     

Functional Consequences of Changing Proline Residues in the Phenylalanine-Specific Permease of Escherichia coli

The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on genetic analysis involving the construction of protein fusions with alkaline phosphatase has previously been proposed in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm (J. Pi and A. J. Pittard, J. Bacteriol. 178:2650–2655, 1996). Site-directed mutagenesis has been used to investigate the functional importance of each of the 16 proline residues of the PheP protein. Replacement of alanine at only three positions, P54, P341, and P442, resulted in the loss of 50% or more activity. Substitutions at P341 had the most dramatic effects. None of these changes in transport activity were, however, associated with any defect of the mutant protein in inserting into the membrane, as indicated by [³⁵S]methionine labelling and immunoprecipitation using anti-PheP serum. A possible role for each of these three prolines is discussed. Inserting a single alanine residue at different sites within span IX and the loop immediately preceding it also had major effects on transport activity, suggesting an important role for a highly organized structure in this region of the protein.