Engineered viruses to select genes encoding secreted and membrane-bound proteins in mammalian cells

We have developed a functional genomics tool to identify the subset of cDNAs encoding secreted and membrane-bound proteins within a library (the ‘secretome’). A Sindbis virus replicon was engineered such that the envelope protein precursor no longer enters the secretory pathway. cDNA fragments were fused to the mutant precursor and expression screened for their ability to restore membrane localization of envelope proteins. In this way, recombinant replicons were released within infectious viral particles only if the cDNA fragment they contain encodes a secretory signal. By using engineered viral replicons to selectively export cDNAs of interest in the culture medium, the methodology reported here efficiently filters genetic information in mammalian cells without the need to select individual clones. This adaptation of the ‘signal trap’ strategy is highly sensitive (1/200 000) and efficient. Indeed, of the 2546 inserts that were retrieved after screening various libraries, more than 97% contained a putative signal peptide. These 2473 clones encoded 419 unique cDNAs, of which 77% were previously annotated. Of the 94 cDNAs encoding proteins of unknown function, 24% either had no match in databases or contained a secretory signal that could not be predicted from electronic data.     

Therapeutic efficacy of melanoma-reactive TIL injected in stage III melanoma patients

Adoptive therapy for cancer using tumor-infiltrating lymphocytes (TIL) has mainly been investigated in cancer patients with advanced stage disease. The limited clinical success has not been encouraging, although this might be explained by poor TIL specificity and/or high tumor burden. To re-evaluate the effectiveness of adoptive therapy, we analyzed the capacity of tumor-reactive TIL injection in preventing the further development of disease in stage III melanoma patients after complete tumor resection. A phase II/III randomized trial was performed on 88 melanoma patients, who received autologous TIL plus interleukin-2 (IL-2) or IL-2 only. The duration of relapse-free survival was analyzed, taking into account the immunological specificity of injected TIL and the number of metastatic lymph nodes removed before treatment. Kaplan-Meyer analysis revealed that the injection of tumor-reactive TIL was statistically correlated with prolonged relapse-free survival in patients with only one metastatic lymph node. Therefore, improved clinical outcome could be obtained after adoptive therapy by selecting appropriate groups of patients and monitoring the specificity of the injected TIL populations.     

Structural evidence of functional divergence in human alkaline phosphatases

The evolution of the alkaline phosphatase (AP) gene family has lead to the existence in humans of one tissue-nonspecific (TNAP) and three tissue-specific isozymes, i.e. intestinal (IAP), germ cell (GCAP), and placental AP (PLAP). To define the structural differences between these isozymes, we have built models of the TNAP, IAP, and GCAP molecules based on the 1.8-structure of PLAP(1) and have performed a comparative structural analysis. We have examined the monomer-monomer interface as this area is crucial for protein stability and enzymatic activity. We found that the interface allows the formation of heterodimers among IAP, GCAP, and PLAP but not between TNAP with any of the three tissue-specific isozymes. Secondly, the active site cleft was mapped into three regions, i.e. the active site itself, the roof of the cleft, and the floor of the cleft. This analysis led to a structural fingerprint of the active site of each AP isozyme that suggests a diversification in substrate specificity for this isozyme family.     

Inflammatory reaction and changes in expression of coagulation proteins on lung endothelial cells after total-body irradiation in mice

Inflammatory reaction is a classical feature of radiation exposure, and pneumonitis is a dose-limiting complication in the handling of hematological disorders treated with total-body irradiation. In the present study, we first evaluated the inflammatory response in C57BL6/J mice exposed to lethal doses of gamma rays treated with antibiotics or not. Both interleukin 6 and KC (also known as Gro1) were increased in the plasma 10 to 18 days after radiation exposure, independent of bacterial infection, whereas fibrinogen release was linked to a bacterial infection. Furthermore, both Il6 and KC were increased in the lungs of irradiated mice. Our second objective was to characterize the endothelial cell changes in the lungs of total-body-irradiated mice. For this purpose, a quantitative RT-PCR was used to determine the expression of genes involved in inflammatory and coagulation processes. We found that the adhesion molecules P-selectin and platelet endothelial cell adhesion molecule 1 were up-regulated, whereas E-selectin remained unchanged. Tissue factor expression was up-regulated as well, and thrombomodulin gene expression was down-regulated. The investigation by immunohistochemistry of adhesion molecules confirmed the increase in the basal expression of both P-selectin and platelet endothelial cell adhesion molecule 1 on pulmonary endothelial cells. All together, our results suggest the involvement of endothelial cells in the development of radiation-induced inflammatory and thrombotic processes.     

Comparison of year-of-exam- and age-matched estimates of heritability in the Framingham Heart Study data

Several different approaches can be used to examine generational and temporal trends in family studies. The measurement of offspring and parents can be made over a short period of time with parents and offspring having quite different ages, or measurements can be made at the same ages but with decades between parent and offspring measures. A third approach, used in the Framingham Heart Study, has repeated examinations across a broad range of age and time, and provides a unique opportunity to compare these approaches. Parents and offspring were matched both on (year of exam) and on age. Heritability estimates for systolic blood pressure, body mass index, height, weight, cholesterol, and glucose were obtained by regressing offspring on midparent values with and without adjustment for age. Higher estimates of heritability were obtained for age-matched than for year-of-exam-matched data for all traits considered. For most traits, estimates of the heritability of the change over time (slope) of the trait were near zero. These results suggest that the optimal design to identify genetic effects in traits with large age-related effects may be to measure parents and offspring at similar ages and not to rely on age-adjustment or longitudinal measures to account for these temporal effects.     

Impact of Legume Flour Addition on Pasta Structure: Consequences on Its <Emphasis Type="Italic">In Vitro</Emphasis> Starch Digestibility

Pasta is popular for its ease of cooking and its low glycaemic index (GI). This interesting nutritional property can be attributed to its specific compact structure generally described as a protein network entrapping starch granules. Despite this low GI, pasta is poor in fibres and lack some essential amino acids. To enhance its nutritional composition, pasta can be fortified with non-traditional ingredients such as legume flours. The objective of this study was to investigate the impact of legume flour addition on pasta structure and the inherent consequences on the in vitro digestibility of starch. The addition of a high level (35%, w/w) of legume flour, especially split pea flour, induced some minor structural changes in pasta. The inclusion of fibres, the dilution of gluten proteins by albumins and globulins, and the larger amount of thin protein films (in split pea pasta) may have favoured higher susceptibility of starch to digestive enzymes. At the opposite, the presence of some partially gelatinised starch granules in the core of fortified pasta may have favoured the decrease in the in vitro starch digestibility. As a consequence, a high level of legume flour addition in pasta did not have any significant impact on its in vitro starch digestibility. A high level of split pea and faba bean flours can thus be added to pasta to increase its nutritional composition while keeping its low glycaemic index.     

Gender affects liver desaturase expression in a rat model of n−3 fatty acid repletion

Dietary n?3 polyunsaturated fatty acids (PUFA) are major components of cell membranes and have beneficial effects on human health. Docosahexaenoic acid (DHA; 22:6n?3) is the most biologically important n?3 PUFA and can be synthesized from its dietary essential precursor, α-linolenic acid (ALA; 18:3n?3). Gender differences in the efficiency of DHA bioconversion have been reported, but underlying molecular mechanisms are unknown. We compared the capacity for DHA synthesis from ALA and the expression of related enzymes in the liver and cerebral cortex between male and female rats. Wistar rats, born with a low-DHA status, were supplied with a suboptimal amount of ALA from weaning to 8 weeks of age. Fatty acid composition was determined by gas chromatography, the mRNA expression of different genes involved in PUFA metabolism was determined by RT-PCR (low-density array) and the expression of proteins was determined by Western blot analysis. At 8 weeks, DHA content was higher (+20 to +40%) in each phospholipid class of female livers compared to male livers. The “Δ4,” Δ5 and Δ6 desaturation indexes were 1.2–3 times higher in females than in males. The mRNA expression of Δ5- and Δ6-desaturase genes was 3.8 and 2.5 times greater, respectively, and the Δ5-desaturase protein was higher in female livers (+50%). No gender difference was observed in the cerebral cortex. We conclude that female rats replete their DHA status more readily than males, probably due to a higher expression of liver desaturases. Our results support the hypothesis on hormonal regulation of PUFA metabolism, which should be taken into account for specific nutritional recommendations.     

New insights into the membrane topology of the phagocyte NADPH oxidase: characterization of an anti-gp91-phox conformational monoclonal antibody

Cytochrome b(558) is the catalytic core of the phagocyte NADPH oxidase that mediates the production of bactericidal reactive oxygen species. Cytochrome b(558) is formed by two subunits gp91-phox and p22-phox (1/1), non-covalently associated. Its activation depends on the interaction with cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac) leading to an electron transfer from NADPH to molecular oxygen and to the release of superoxide anions. Several studies have suggested that the activation process was linked to a change in cytochrome b(558) conformation. Recently, we confirmed this hypothesis by isolating cytochrome b(558) in a constitutively active form. To characterize active and inactive cytochrome b(558) conformations, we produced four novel monoclonal antibodies (7A2, 13B6, 15B12 and 8G11) raised against a mixture of cytochrome b(558) purified from both resting and stimulated neutrophils. The four antibodies labeled gp91-phox and bound to both native and denatured cytochrome b(558). Interestingly, they were specific of extracellular domains of the protein. Phage display mapping combined to the study of recombinant gp91-phox truncated forms allowed the identification of epitope regions. These antibodies were then employed to investigate the NADPH oxidase activation process. In particular, they were shown to inhibit almost completely the NADPH oxidase activity reconstituted in vitro with membrane and cytosol. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils pointed out the capacity of the monoclonal antibody 13B6 to bind preferentially to the active form of cytochrome b(558). All these data suggested that the four novel antibodies are potentially powerful tools to detect the expression of cytochrome b(558) in intact cells and to analyze its membrane topology. Moreover, the antibody 13B6 may be conformationally sensitive and used as a probe for identifying the active NADPH oxidase complex in vivo.     

Design, synthesis, and antimalarial activity of structural chimeras of thiosemicarbazone and ferroquine analogues

The design, synthesis, and antimalarial activity of chimeras of thiosemicarbazones (TSC) and ferroquine (FQ) is reported. Key structural elements derived from FQ were coupled to fragments capable of coordinating metal ions. Biological evaluation was conducted against four strains of the malaria parasite Plasmodium falciparum and against the parasitic cysteine protease falcipain-2. To establish the role of the ferrocenyl moiety in the antiplasmodial activity of this series, purely organic parent compounds were also synthesized and tested. The presence of the aminoquinoline structure, allowing transport of the compounds to the food vacuole of the parasite, seems to be the major contributor to antimalarial activity.     

Mammalian cilia function is independent of the polymeric state of tubulin glycylation

Polyglycylation is a polymeric post-translational modification of tubulin that is ubiquitous and widely present in cilia and flagella. It consists of the addition of highly variable numbers of glycyl residues as side chains onto the gamma carboxyl group of specific glutamyl residues at the C-termini of alpha- and beta-tubulin. The function of polyglycylation is poorly understood, however, studies in Tetrahymena have shown that the mutation of polyglycylation sites in beta-tubulin resulted in axonemal abnormality or lethality. This suggests that polyglycylation is functionally essential in protists. We hypothesize that polyglycylation is also essential in mammalian cilia and that the extent of polyglycylation has functional significance. In this study, we examined polyglycylation states in ciliated tissues and in mouse tracheal epithelial cell cultures. We utilized two antibodies, TAP 952 and AXO 49, which recognize glutamyl sites possessing monomeric glycylation sites and glutamyl sites possessing polymeric glycylation sites, respectively. Monomeric glycylation sites were observed in cilia of all the ciliated tissues examined but were invariably excluded from the distal tips. In contrast, polymeric glycylation sites were rare, but when observed, they were localized at the bases of cilia. During ciliogenesis, in epithelial cell cultures, monomeric glycylation sites were observed, but the extent of polymeric glycylation sites were variable and were only observed during the early stages of the cultures. Our observations suggest that while monomeric glycylation sites are universal and likely essential in mammalian cilia, polymeric glycylation sites are not required for ciliary beating. Rather, our observations suggest that the number of added glycyl residues increases progressively from the tips of cilia toward their bases.