Microanalysis of plant mitochondrial protein synthesis products:

Summary A mitochondrial fraction obtained from 0.5 g of leaves was purified on a 0.75 ml Percoll gradient and used for an in vitro mitochondrial protein synthesis assay in the presence of [³⁵S] methionine. A set of 15 to 20 labeled polypeptides were revealed by autoradiography after sodium dodecylsulfate polyacrylamide gel electrophoresis. This could be applied at an early growth stage by using a few leaves from individual seedlings. It revealed the presence of variant mitochondrially translated polypeptides in green leaves of cytoplasmic male sterile lines from various cultivated plants of large economic importance: maize, wheat, sugar beet, tobacco and faba bean. This non-destructive microanalysis is thus of general use and opens new possibilities for rapid and large mass screening of mitochondrial parameters such as male sterility.     

Isosorbide dinitrate bioconversion by Beauveria strains: implication of glutathione transferase levels

Summary The level of glutathione S-transferase (GSH0ST) activity was determined in growing cultures and in washed resting cells of Beauveria strains with and without addition of isosorbide dinitrate (ISDN), by following the reaction with o-dinitrobenzene (o-DNB). The level of GSH-ST varied according to the pH changes of the medium and decreased during culture. The enzymatic activity measured with o-DNB did not correlate with ISDN bioconversion carried out either with B. sulfurescens or B. tenella. Immediately after starting incubation of the resting cells with ISDN, the level of GSH-ST activity initially increased, but declined afterwards, whereas the bioconversion process continued and reached 500 mg/l isosorbide 5-mononitrate. When 1-chloro-2,4-dinitrobenzene was used as a substrate for the evaluation of GSH-ST activity using B. tenella, a conjugation product having a UV absorption at 410 nm was formed.     

A virus-specific CD4+ cell-mediated cytolytic activity revealed by CD8+ cell elimination regularly develops in uncloned human antiviral cell lines

Antiviral HLA class II-restricted cytotoxic CD4+ clones have been relatively well characterized in vitro but their significance in the immune response remains unknown. Here anti-influenza A and anti-EBV CD4+ CTL have been studied by using permanent cell lines either untreated or depleted of CD8+ cells. In bulk cultures, HLA class I-restricted anti-viral CD8+ CTL account for all of the detectable killer cell activity, whereas after elimination of CD8+ cells an HLA class II-restricted killer activity mediated by CD4+/2H4-/4B4+ cells was consistently observed. The CD4+ CTL were fully differentiated in all of the cultures tested from the third in vitro passage because they could be demonstrated immediately after elimination of CD8+ cells. These CD4+ killer cells were equivalent to the CD8+ cells in terms of their lytic capacity. The absence of any class II-restricted antiviral activity in bulk cultures seems to be related to the very small numbers of CD4+ cells present in these antiviral cell lines. However, CD4+ cytolytic activity could not be detected during the first two in vitro passages, even when limiting dilution analysis of the CTL precursors were performed, showing that the killer function of Th cells differentiate only after several in vitro stimulations.     

Three epitopic peptides of the simian immunodeficiency virus Nef protein recognized by macaque cytolytic T lymphocytes.

In 8 of 12 experimentally infected macaques, the Nef SIVmac 251 protein was recognized by cytolytic T lymphocytes (CTL) and appeared strongly immunogenic. Here, we report experiments which have been performed by using synthetic peptides to precisely determine the epitopes recognized by macaque CTL. Three epitopes of the Nef protein have been defined as CTL targets in three macaques. The epitopic peptides are located in the central region of the protein, and all of them show high homology with peptides of the human immunodeficiency virus type 1 Nef protein recognized by human CTL in association with several human leukocyte antigen molecules. These results suggest that (i) the Nef protein is a good candidate for vaccination not only because of its early expression but also because of its high immunogenicity for CTL, (ii) long peptides covering the central region of this protein could be used as vaccines and could cross the major histocompatibility complex barrier in a large variety of individuals, and (iii) the rhesus macaque is a good animal model in which to test for protection by CTL.     

ROP18 is a rhoptry kinase controlling the intracellular proliferation of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite for which the discharge of apical organelles named rhoptries is a key event in host cell invasion. Among rhoptry proteins, ROP2, which is the prototype of a large protein family, is translocated in the parasitophorous vacuole membrane during invasion. The ROP2 family members are related to protein-kinases, but only some of them are predicted to be catalytically active, and none of the latter has been characterized so far. We show here that ROP18, a member of the ROP2 family, is located in the rhoptries and re-localises at the parasitophorous vacuole membrane during invasion. We demonstrate that a recombinant ROP18 catalytic domain (amino acids 243-539) possesses a protein-kinase activity and phosphorylate parasitic substrates, especially a 70-kDa protein of tachyzoites. Furthermore, we show that overexpression of ROP18 in transgenic parasites causes a dramatic increase in intra-vacuolar parasite multiplication rate, which is correlated with kinase activity. Therefore, we demonstrate, to our knowledge for the first time, that rhoptries can discharge active protein-kinases upon host cell invasion, which can exert a long-lasting effect on intracellular parasite development and virulence.     

Inverted topology of the Toxoplasma gondii ROP5 rhoptry protein provides new insights into the association of the ROP2 protein family with the parasitophorous vacuole membrane

Toxoplasma gondii, as many intracellular parasites, is separated from the cytosol of its host cell by a parasitophorous vacuole membrane (PVM). This vacuole forms during host cell invasion and parasite apical organelles named rhoptries discharge proteins that associate with its membrane during this process. We report here the characterization of the rhoptry protein ROP5, which is a new member of the ROP2 family. Contrasting with what is known for other ROP2 family proteins, ROP5 is not processed during trafficking to rhoptries. We show here that ROP5 is secreted during invasion and associates with the PVM. Using differential permeabilization of infected cells, we have shown that ROP5 exposes its C-terminus towards the host cell cytoplasm, which corresponds to a reverse topology compared with ROP2 and ROP4. Taken together with recent modelling data suggesting that the C-terminal hydrophobic domain hitherto described as transmembrane may correspond to a hydrophobic helix buried in the catalytic domain of kinase-related proteins, these findings call for a reappraisal of the current view of ROP2 family proteins association with the PVM.     

LYST Controls the Biogenesis of the Endosomal Compartment Required for Secretory Lysosome Function

Chediak–Higashi syndrome (CHS) is caused by mutations in the gene encoding LYST protein, the function of which remains poorly understood. Prominent features of CHS include defective secretory lysosome exocytosis and the presence of enlarged, lysosome‐like organelles in several cell types. In order to get further insight into the role of LYST in the biogenesis and exocytosis of cytotoxic granules, we analyzed cytotoxic T lymphocytes (CTLs) from patients with CHS. Using confocal microscopy and correlative light electron microscopy, we showed that the enlarged organelle in CTLs is a hybrid compartment that contains proteins components from recycling‐late endosomes and lysosomes. Enlargement of cytotoxic granules results from the progressive clustering and then fusion of normal‐sized endolysosomal organelles. At the immunological synapse (IS) in CHS CTLs, cytotoxic granules have limited motility and appear docked while nevertheless unable to degranulate. By increasing the expression of effectors of lytic granule exocytosis, such as Munc13‐4, Rab27a and Slp3, in CHS CTLs, we were able to restore the dynamics and the secretory ability of cytotoxic granules at the IS. Our results indicate that LYST is involved in the trafficking of the effectors involved in exocytosis required for the terminal maturation of perforin‐containing vesicles into secretory cytotoxic granules.      

Do aphids actively search for ant partners?

The aphid–ant mutualistic relationships are not necessarily obligate for neither partners but evidence is that such interactions provide them strong advantages in terms of global fitness. While it is largely assumed that ants actively search for their mutualistic partners namely using volatile cues; whether winged aphids (i.e., aphids’ most mobile form) are able to select ant‐frequented areas had not been investigated so far. Ant‐frequented sites would indeed offer several advantages for these aphids including a lower predation pressure through ant presence and enhanced chances of establishing mutuaslistic interactions with neighbor ant colonies. In the field, aphid colonies are often observed in higher densities around ant nests, which is probably linked to a better survival ensured by ants’ services. Nevertheless, this could also result from a preferential establishment of winged aphids in ant‐frequented areas. We tested this last hypothesis through different ethological assays and show that the facultative myrmecophilous black bean aphid, Aphis fabae L., does not orientate its search for a host plant preferentially toward ant‐frequented plants. However, our results suggest that ants reduce the number of winged aphids leaving the newly colonized plant. Thus, ants involved in facultative myrmecophilous interactions with aphids appear to contribute to structure aphid populations in the field by ensuring a better establishment and survival of newly established colonies rather than by inducing a deliberate plant selection by aphid partners based on the proximity of ant colonies.