Metabolic control of seed germination

We have used proteomics to better characterize germination and early seedling vigor in sugarbeet. Our strategy includes (1) construction of proteome reference maps for dry and germinating seeds of a high-vigor reference seed lot; (2) investigation of the specific tissue accumulation of proteins (root, cotyledon, perisperm); (3) investigation of changes in protein expression profiles detected in the reference seed lot subjected to different vigor-modifying treatments, e.g. aging and/or priming. More than 1 000 sugarbeet seed proteins have been identified by LC/MS-MS mass spectrometry (albumins, globulins and glutelins have been analyzed separately). Due to the conservation of protein sequences and the quality of MS sequencing (more than 10 000 peptide sequences have been obtained), the success rate of protein identification was on the average of 80%. This is to our knowledge the best detailed proteome analysis ever carried out in seeds. The data allowed us to build a detailed metabolic chart of the sugarbeet seed, generating new insights into the molecular mechanisms determining the development of a new seedling. Also, the proteome of a seed-storage tissue as the perisperm is described for the first time.     

Interleukin-17A mediates acquired immunity to pneumococcal colonization

Although anticapsular antibodies confer serotype-specific immunity to pneumococci, children increase their ability to clear colonization before these antibodies appear, suggesting involvement of other mechanisms. We previously reported that intranasal immunization of mice with pneumococci confers CD4+ T cell-dependent, antibody- and serotype-independent protection against colonization. Here we show that this immunity, rather than preventing initiation of carriage, accelerates clearance over several days, accompanied by neutrophilic infiltration of the nasopharyngeal mucosa. Adoptive transfer of immune CD4+ T cells was sufficient to confer immunity to na?ve RAG1(-/-) mice. A critical role of interleukin (IL)-17A was demonstrated: mice lacking interferon-gamma or IL-4 were protected, but not mice lacking IL-17A receptor or mice with neutrophil depletion. In vitro expression of IL-17A in response to pneumococci was assayed: lymphoid tissue from vaccinated mice expressed significantly more IL-17A than controls, and IL-17A expression from peripheral blood samples from immunized mice predicted protection in vivo. IL-17A was elicited by pneumococcal stimulation of tonsillar cells of children or adult blood but not cord blood. IL-17A increased pneumococcal killing by human neutrophils both in the absence and in the presence of antibodies and complement. We conclude that IL-17A mediates pneumococcal immunity in mice and probably in humans; its elicitation in vitro could help in the development of candidate pneumococcal vaccines.     

The Transmembrane Domain of BST-2 Determines Its Sensitivity to Down-Modulation by Human Immunodeficiency Virus Type 1 Vpu

Bone marrow stromal cell antigen 2 (BST-2, also known as tetherin) restricts the production of a number of enveloped viruses by blocking virus release from the cell surface. This antiviral activity is counteracted by such viral factors as Vpu of human immunodeficiency virus type 1 (HIV-1). Here, we report that Vpu antagonizes human BST-2 but not BST-2 derived from African green monkeys. The determinants of susceptibility to Vpu map to the transmembrane domain of BST-2. In accordance with this, expression of human BST-2 containing a modified transmembrane domain effectively blocks the replication of wild-type Vpu-expressing HIV-1 in CD4⁺ T cells. Furthermore, these BST-2 variants, as opposed to wild-type human BST-2, are refractory to Vpu-mediated down-regulation as a result of an attenuated interaction with Vpu. In view of the work by others pointing to a key role of the transmembrane domain of Vpu in promoting virus release, our data suggest that a direct interaction through the transmembrane domain of each of these two proteins is a prerequisite for Vpu to down-modulate BST-2.Human immunodeficiency virus type 1 (HIV-1) encodes four accessory proteins, Vif, Vpr, Vpu, and Nef. Although they are dispensable for HIV-1 replication in certain transformed cell lines, these accessory proteins play important roles in HIV-1 pathogenesis by modulating host immunity and overcoming antagonism by cellular factors (10). For example, Vif counteracts APOBEC3G by recruiting the cullin 5-elongin B/C ubiquitin ligase complex and sending polyubiquitinated APOBEC3G to proteasomes for degradation (29). In the absence of Vif, newly synthesized APOBEC3G is incorporated into virus particles and hampers the production of infectious proviral DNA in the new round of infection (4, 10, 23). In addition to its role in down-modulating the cell surface expression of CD4 in infected T cells (11), Vpu stimulates HIV-1 production in cells such as HeLa cells (26). The mechanism behind this latter activity of Vpu was unknown until it was recently discovered that bone marrow stromal cell antigen 2 (BST-2, also known as tetherin, CD317, or HM1.24) blocks the release of HIV-1 and that this inhibitory effect is antagonized by viral Vpu (16, 25).BST-2 harbors an N-terminal transmembrane domain and a C-terminal glycosyl-phosphatidylinositol anchor that together create an unusual topology with both termini of BST-2 inserted into the plasma membrane (8, 18). This unique topology of BST-2 may underlie the mechanism for the retention of progeny virus particles at the cell surface (16). An indirect mechanism behind this tethering effect has not been ruled out, especially in view of the difficulty of detecting BST-2 protein in purified HIV-1 particles (14). In addition to HIV-1, a number of enveloped viruses are subject to inhibition by BST-2, including simian immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, Mason-Pfizer monkey virus, and Lassa virus, as well as Ebola and Marburg viruses (5, 6, 16, 19, 25). This suggests that BST-2 has a broad antiviral effect spectrum.The bst-2 gene has in its promoter the IRF-1/2 and ISGF3 response elements and thus belongs to the interferon-stimulated gene family (17). In line with its ability to impair the release of enveloped viruses, BST-2 has been demonstrated to be the effector in human embryonic kidney (HEK293T) cells that leads to the interferon-induced block of Vpu deletion-containing HIV-1 production (15). However, the African green monkey kidney cell line COS-7 responds to interferon treatment with a different outcome in that the production of both Vpu deletion-containing and Vpu-expressing HIV-1 is inhibited (15). This indicates that interferon induces a block to HIV-1 in COS-7 cells that cannot be overcome by Vpu. A conceivable candidate that creates this block is BST-2 in COS-7 cells (hereafter named agmBST-2). In this study, we provide evidence that depletion of endogenous BST-2 in COS-7 cells greatly alleviates interferon-induced inhibition of HIV-1 production. The refractoriness of agmBST-2 to Vpu results from a weak association of these two proteins and a resistance of agmBST-2 to Vpu-mediated down-regulation.     

Gender-dependent regulation of G-protein-gated inwardly rectifying potassium current in dorsal raphe neurons in knock-out mice devoid of the 5-hydroxytryptamine transporter

Agonists at G-protein-coupled receptors in neurons of the dorsal raphe nucleus (DRN) of knock-out mice devoid of the serotonin transporter (5-HTT(-/-)) exhibit lower efficacy to inhibit cellular discharge than in wild-type counterparts. Using patch-clamp whole-cell recordings, we found that a G-protein-gated inwardly rectifying potassium (GIRK) current is involved in the inhibition of spike discharge induced by 5-HT1A agonists (5-carboxamidotryptamine (5-CT) and (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (8-OH-DPAT); 50 nM-30 microM) in both wild-type and 5-HTT(-/-) female and male mice. These effects were mimicked by 5'-guanylyl-imido-diphosphate (Gpp(NH)p; 400 microM) dialysis into cells with differences between genders. The 5-HTT(-/-) knock-out mutation reduced the current density induced by Gpp(NH)p in females but not in males. These data suggest that the decreased response of 5-HT1A receptors to agonists in 5-HTT(-/-) mutants reflects notably alteration in the coupling between G-proteins and GIRK channels in females but not in males. Accordingly, gender differences in central 5-HT neurotransmission appear to depend-at least in part-on sex-related variations in corresponding receptor-G protein signaling mechanisms.     

Direct gene transfer inPetunia hybrida electroporated protoplasts: Evidence for co-transformation with a phosphoenolpyruvate carboxylase cDNA from sorghum leaf

Summary Co-transformation experiments were carried out onPetunia hybrida protoplasts. The method used was electroporation with two plasmids: one confering kanamycin resistance, and the other harbouring a phosphoenolpyruvate carboxylase (PEPC) cDNA fromSorghum vulgare leaves. Southern blot analysis of the selected lines demonstrated a high co-transformation frequency.Abbreviations PEPC phosphoenolpyruvate carboxylase - NPT neomycin phosphotransferase - ATF absolute transformation frequency - PEG polyethylenglycol - BA 6-benzyl-aminopurine - IAA 3-indoleacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Relation between protoplast division,cell-cycle stage and nuclear chromatin structure

Summary Using different sources of protoplasts and two complementary techniques, flow cytometry and image analysis, to study the cell-cycle phases, we sought to define the particular protoplast state associated with the disposition to divide. Both inPetunia and inNicotiana plumbaginifolia, tissues with a higher G2 frequency (from different aged plants) yielded protoplasts capable of increased cell division. InSorghum, the age of the plant does not modify the proportion of G2 nuclei in leaf protoplasts, and we used root protoplasts to increase G2 frequencies. InHelianthus annuus, leaf protoplasts did not divide; however, hypocotyl protoplast preparations with relatively high 4C DNA frequencies do divide. Moreover, image analysis of chromatin structure indicated that leaf nuclei were in the G0 phase, unlike those from hypocotyls which were in G1. A high frequency of protoplasts with G2 nuclei appears to be correlated with the ability of a given preparation to undergo division; conversely, the differentiated G0 state is not conducive to division.