Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis

BACKGROUND: Embryonic cleavage leads to the formation of an epithelial layer during development. In Drosophila, the process is specialized and called cellularization. The trafficking pathways that underlie this process and that are responsible for the mobilization of membrane pools, however, remain poorly understood. RESULTS: We provide functional evidence for the role of endocytic trafficking through Rab11 endosomes in remobilizing vesicular membrane pools to ensure lateral membrane growth. Part of the membrane stems from endocytosed apical material. Mutants in the endocytic regulators rab5 and shibire/dynamin inhibit basal-lateral membrane growth, and apical endocytosis is blocked in shibire mutants. In addition, shibire controls vesicular trafficking through Rab11-positive endosomes. In shibire mutants, the transmembrane protein Neurotactin follows the secretory pathway normally but is not properly inserted in the plasma membrane and accumulates instead in Rab11 subapical endosomes. Consistent with a direct role of shibire in vesicular trafficking through Rab11 endosomes, Shibire is enriched in this compartment. Moreover, we show by electron microscopy the large accumulation of intracellular coated pits on subapical endocytic structures in shibire mutants. Finally, we show that Rab11 is essential for membrane growth and invagination during cellularization. CONCLUSION: Together, the data show that endocytic trafficking is required for basal-lateral membrane growth during cellularization. We identify Rab11 endosomes as key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization. This pathway may be required in other morphogenetic processes characterized by the growth of a membrane domain.     

HER-2 DNA and protein vaccines containing potent Th cell epitopes induce distinct protective and therapeutic antitumor responses in HER-2 transgenic mice

Overexpression of the growth factor receptor HER-2 (c-erbB-2, neu) has transforming potential and occurs in approximately 20-30% of breast and ovarian cancers. HER-2 is a self Ag, but Abs and T cells specific for HER-2 have been isolated from cancer patients, suggesting HER-2 may be a good target for active immunotherapy. We constructed rat HER-2 DNA and protein vaccines containing potent Th cell epitopes derived from tetanus toxin and studied their potency in two strains of mice transgenic for the rat HER-2 molecule. Vaccination with HER-2 DNA protected nontransgenic mice from tumor challenge, but induced only moderate protection in one of the tumor models. However, vaccination with the modified HER-2 protein resulted in almost complete protection from tumor challenge in both tumor models. This protection could be mediated by Abs alone. In addition, protein vaccination efficiently eliminated pre-established tumors in both models, even when vaccination occurred 9 days after tumor implantation. These data demonstrate the potential of HER-2-based vaccines as therapeutic agents for the treatment of cancers overexpressing HER-2.     

Deriving topological constraints from functional data for the design of reagentless fluorescent immunosensors

The possibility of obtaining, from any antibody, a fluorescent conjugate which responds to the binding of the antigen by a variation of fluorescence, would be of great interest in the micro- and nano-analytical sciences. This possibility was explored with antibody mAb4E11, which is directed against the dengue virus and for which no structural data is available. Three rules of design were developed to identify residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by mutagenesis. (i) The target residue belonged to the hypervariable loops of the antibody. (ii) It was adjacent, along the amino acid sequence of the antibody, to a residue which was functionally important for the interaction with the antigen. (iii) It was not important in itself for the interaction with the antigen. Eight conjugates between a single chain variable fragment of mAb4E11 and an environment-sensitive fluorophore were constructed. Three of them showed an increase in their fluorescence intensity by 1.5-2.8-fold on antigen binding, without loss of affinity. This increase allowed the titration of the antigen in serum above a threshold concentration of 10nM. Experiments of quenching with potassium iodide suggested that the fluorescence variation was due to a shielding of the fluorescent group from the solvent by the binding of the antigen, and that therefore its mechanism is general.     

Functional and structural basis of carnitine palmitoyltransferase 1A deficiency

Carnitine palmitoyltransferase 1A (CPT1A) is the key regulatory enzyme of hepatic long-chain fatty acid beta-oxidation. Human CPT1A deficiency is characterized by recurrent attacks of hypoketotic hypoglycemia. We presently analyzed at both the functional and structural levels five missense mutations identified in three CPT1A-deficient patients, namely A275T, A414V, Y498C, G709E, and G710E. Heterologous expression in Saccharomyces cerevisiae permitted to validate them as disease-causing mutations. To gain further insights into their deleterious effects, we localized these mutated residues into a three-dimensional structure model of the human CPT1A created from the crystal structure of the mouse carnitine acetyltransferase. This study demonstrated for the first time that disease-causing CPT1A mutations can be divided into two categories depending on whether they affect directly (functional determinant) or indirectly the active site of the enzyme (structural determinant). Mutations A275T, A414V, and Y498C, which exhibit decreased catalytic efficiency, clearly belong to the second class. They are located more than 20 A away from the active site and mostly affect the stability of the protein itself and/or of the enzyme-substrate complex. By contrast, mutations G709E and G710E, which abolish CPT1A activity, belong to the first category. They affect Gly residues that are essential not only for the structure of the hydrophobic core in the catalytic site, but also for the chain-length specificity of CPT isoforms. This study provides novel insights into the functionality of CPT1A that may contribute to the design of drugs for the treatment of lipid disorders.     

Plasmalogens protect unsaturated lipids against UV-induced oxidation in monolayer

Oxidative stress results from the attack by free radicals of several cellular targets (proteins, DNA and lipids). The cell equilibrium is a direct consequence of the pro-/antioxidant balance. In order to understand the physiological processes involved in oxidative stress, we followed oxidation of unsaturated lipids using a biomimetic system: Langmuir monolayers. The oxidation mode chosen was UV-irradiation and the lipid model was a polyunsaturated phospholipid: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC). The monomolecular film technique was used to measure membrane rheology before and after UV-irradiation. We showed that the UV-irradiation of a DLPC monomolecular film led to a molecular area and surface elasticity modulus decrease that attests to the apparition of new molecular species at the air-water interface. The antioxidant effect of a synthetic plasmalogen (1-O-(1'-(Z)-hexadecenyl)-2-O-oleoyl-sn-glycero-3-phosphocholine or P(PLM)OPE) was tested on the oxidation of DLPC. Indeed, for about 25% mol P(PLM)OPE in mixed DLPC/P(PLM)OPE monolayers, a complete inhibition of the molecular area and the surface elasticity modulus decreases was observed in our experimental conditions. Lower P(PLM)OPE quantities delayed but did not prevent the DLPC oxidation in mixed monolayers.     

Origin and diffusion of mtDNA haplogroup X

A maximum parsimony tree of 21 complete mitochondrial DNA (mtDNA) sequences belonging to haplogroup X and the survey of the haplogroup-associated polymorphisms in 13,589 mtDNAs from Eurasia and Africa revealed that haplogroup X is subdivided into two major branches, here defined as “X1” and “X2.” The first is restricted to the populations of North and East Africa and the Near East, whereas X2 encompasses all X mtDNAs from Europe, western and Central Asia, Siberia, and the great majority of the Near East, as well as some North African samples. Subhaplogroup X1 diversity indicates an early coalescence time, whereas X2 has apparently undergone a more recent population expansion in Eurasia, most likely around or after the last glacial maximum. It is notable that X2 includes the two complete Native American X sequences that constitute the distinctive X2a clade, a clade that lacks close relatives in the entire Old World, including Siberia. The position of X2a in the phylogenetic tree suggests an early split from the other X2 clades, likely at the very beginning of their expansion and spread from the Near East.     

Docking and small angle X-ray scattering studies of purine nucleoside phosphorylase

Docking simulations have been used to assess protein complexes with some success. Small angle X-ray scattering (SAXS) is a well-established technique to investigate protein spatial configuration. This work describes the integration of geometric docking with SAXS to investigate the quaternary structure of recombinant human purine nucleoside phosphorylase (PNP). This enzyme catalyzes the reversible phosphorolysis of N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for PNP causes gradual decrease in T-cell immunity. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant rejection, rheumatoid arthritis, lupus, and T-cell lymphomas. PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. The present analysis confirms the trimeric structure observed in the crystal. The potential application of the present procedure to other systems is discussed.     

Up-regulation of glucose metabolism during male pronucleus formation determines the early onset of the s phase in bovine zygotes

After in vitro fertilization with spermatozoa from bulls with high in vitro fertility, a beneficial paternal effect is manifested during the G1 phase of the first cell cycle. This benefit determines an earlier onset of the first S phase, and then a successful morula-blastocyst transition 7 days later. We hypothesized that the origin of the paternal effect could be a shift of the metabolism of the fertilized oocyte, because in mice, sperm decondensation is responsible for a dramatic increase in glucose metabolism. In this study we investigated the interaction between both pronuclei and compared glycolysis and pentose phosphate pathway (PPP) activities in bovine oocytes fertilized with spermatozoa from bulls of high or low fertility. Here we demonstrate that male pronucleus formation is necessary for the onset of the S phase in the female pronucleus, and that the component promoting an early S phase in both pronuclei is metabolic and linked to an up-regulation of the PPP during the male pronucleus formation. This long-lasting paternal effect is more evidence of the important role of epigenetic control during early embryo development.     

Nitric oxide inhibits spleen cell proliferative response after burn injury by inducing cytostasis,apoptosis, and necrosis of activated T lymphocytes: role of the guanylate cyclase

We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one.