Synthesis of novel d-glucuronic acid fatty esters using Candida antarctica lipase in tert-butanol

Glucuronic acid n-alkyl esters, a novel class of promising biosurfactants and their corresponding glucose esters with the same side-chain length, were synthesized by direct esterification in a non-aqueous phase (tert-butanol) using an immobilized lipase.     

Vesicular stomatitis virus glycoprotein: a transducing coat for SFV-based RNA vectors

BACKGROUND: Semliki Forest virus (SFV) vectors have a great potential for the induction of protective immunity in a large number of clinical conditions including cancer. Such a potential accounts for the huge efforts made to improve the in vivo expression from SFV vectors. It is noteworthy that efficient in vivo expression strongly relies on the ability to deliver high-titre vectors. To achieve this, the generation of recombinant SFV particles, using independent expression systems for structural SFV genes, has been proposed. However, despite several modifications in the production process, a risk of contamination with replication-competent, or partially recombined, virus has remained. METHODS: Here, we exploit the ability of the vesicular stomatitis virus glycoprotein (VSV-G), expressed in trans, to hijack full-length genomic SFV RNA into secreted virus-like particles (VLPs). To allow SFV vector mobilisation, we designed a CMV driven SFV vector in which the internal 26S promoter has been extensively mutated. With this vector, mobilisation events were monitored using the Green Fluorescent Protein (GFP). The production procedure involves a sequential transfection protocol, of plasmids expressing the VSV-G and the SFV vector respectively. RESULTS: We show that the VLPs are effective for cellular delivery of SFV vectors in a broad range of human and non-human cellular targets. Furthermore, production of VLPs is easy and allows, through concentration, the harvest of high-titre vector. CONCLUSIONS: The present paper describes a convenient process aimed at mobilising full length SFV vectors. A major issue to consider, while developing clinically relevant gene transfer vectors, is the risk of undesirable generation of replication competent by-products. Importantly, as the VSV-G gene shares no homology with the SFV genome, our VLPs offer a strong guarantee of biosafety.     

Large-scale characterization of integral proteins from Arabidopsis vacuolar membrane by two-dimensional liquid chromatography

We developed a method to characterize different classes of membrane proteins within a single experiment and using simple matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. After membrane solubilization with the nondenaturing detergent n-dodecyl-beta-D-maltoside, proteins were separated successively by gel filtration and ion-exchange chromatography and finally by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This procedure allowed to characterize 70 proteins from a membrane fraction enriched in plant vacuolar membrane (Arabidopsis), including integral proteins like the V0 complex of the H(+)-ATPase, the H(+)-pyrophosphatase or the glutathione S-conjugate ATPase AtMRP1, and peripheral proteins like the subunits of the catalytic V1 complex of the H(+)-ATPase. Approximately 60% of identified proteins were predicted to possess at least two trans-membrane domains. Furthermore, proteins, with molecular masses ranging between 20 and 200 kDa were distributed into two populations with maximum frequencies at pI 5.3 and 8.9. Finally, this procedure appeared to allow the identification of proteins known to be minor in whole-cell extracts like signaling or vesicular trafficking proteins. Almost 50% of the proteins identified were functionally unknown whereas the others confirmed that the plant vacuole is a multipurpose compartment.     

Imaging movement of malaria parasites during transmission by Anopheles mosquitoes

Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.     

Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation

Nitric oxide (NO) is synthesized from l-arginine by the Ca(2+)/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca(2+) ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline. Three CaMK II inhibitors, polypeptide 281-302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca(2+)-dependent activation of eNOS.     

Effects of Septal Grafts on Acetylcholine Release from Rat Hippocampus After 192 IgG-Saporin Lesion

The cholinergic inputs to the rat hippocampus were lesioned by intraseptal injections of 192 IgG-saporin. After 15 days, fetal septal cells were grafted into the hippocampus. Thirteen months later, hippocampal acetylcholine (ACh) release was studied by microdialysis. Lesioning reduced basal ACh release (100%) to 20% of normal, which was compensated for by the graft (71%). Infusion of the serotonin uptake inhibitor citalopram (100 M) enhanced ACh release to the same extent (% of basal release) in all rat groups. Systemic injection of 8-OH-DPAT (0.5 mg/kg, SC), an agonist of 5-HT_1A receptors, caused a smaller ACh release than citalopram. Acetylcholinesterase (AChE) staining and densitometric quantification revealed that the lesion-induced reduction of the AChE-staining density was compensated for by septal grafting. In conclusion, both histochemical and biochemical methods showed that cholinergic hippocampal parameters were drastically impaired by 192 IgG-saporin lesions, but were almost completely restored by septal grafting. The graft responded to intrinsic serotonergic regulation.     

PIAS1-mediated sumoylation of focal adhesion kinase activates its autophosphorylation

Focal adhesion kinase (FAK) is a protein tyrosine kinase enriched in focal adhesions, which plays a critical role in integrin-dependent cell motility and survival. The crucial step in its activation is autophosphorylation on Tyr-397, which promotes the recruitment of several enzymes including Src family kinases and the activation of multiple signaling pathways. We found in a yeast two-hybrid screen that the N-terminal domain of FAK interacted with protein inhibitor of activated STAT1 (PIAS1). This interaction was confirmed and shown to be direct using in vitro assays. PIAS1 was co-immunoprecipitated with FAK from transfected cells and brain extracts. PIAS1 has recently been recognized as a small ubiquitin-like modifier (SUMO) ligase. In the presence of PIAS1 and SUMO-1, FAK was sumoylated in intact cells, whereas PYK2, a closely related enzyme, was not. Sumoylation occurred on Lys-152, a residue conserved in FAK during evolution. Sumoylated FAK, like PIAS1, was recovered predominantly from the nuclear fraction. Sumoylation did not require the catalytic activity or autophosphorylation of FAK. In contrast, sumoylation increased dramatically the ability of FAK to autophosphorylate in intact cells and in immune precipitate kinase assays. Endogenous FAK was sumoylated in the presence of PIAS1 and SUMO-1 independently of cell adhesion, and autophosphorylation of sumoylated FAK was persistently increased in suspended cells. These observations show that sumoylation controls the activity of a protein kinase and suggest that FAK may play a novel role in signaling between the plasma membrane and the nucleus.     

Characterization of the membrane-destabilizing properties of different pH-sensitive methacrylic acid copolymers

The intracellular delivery of active biomacromolecules from endosomes into the cytoplasm generally requires a membrane-disrupting agent. Since endosomes have a slightly acidic pH, anionic carboxylated polymers could be potentially useful for this purpose since they can destabilize membrane bilayers by pH-triggered conformational change. In this study, five different pH-sensitive methacrylic acid (MAA) copolymers were characterized with respect to their physicochemical and membrane lytic properties as a function of pH. pH-dependent conformational changes were studied in aqueous solution by turbidimetry and spectrofluorimetry. The hydrophobic domains that formed upon a decrease in pH were found to be dependent on copolymer's composition. Hemolysis and cytotoxicity assays demonstrated that the presence of the hydrophobic ethyl acrylate monomer and/or sufficient protonation of the carboxylic acid groups were important parameters for efficient membrane destabilization. Excessive copolymer hydrophobicity was not associated with membrane destabilization, but resulted in high macrophage cytotoxicity. Overall, this study gave more insights into the structure-activity relationship of MAA copolymers with membrane bilayers. Gaining knowledge of modulation of the physicochemical properties of copolymers and the optimization of copolymer-lipid interactions may lead to the elaboration of much more efficient drug delivery systems.     

Production and targeting of the Brucella abortus antigen L7/L12 in Lactococcus lactis: a first step towards food-grade live vaccines against brucellosis

Brucella abortus is a facultative intracellular gram-negative bacterial pathogen that infects humans and animals by entry mainly through the digestive tract. B. abortus causes abortion in pregnant cattle and undulant fever in humans. The immunogenic B. abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of oral live vaccines against brucellosis, using food-grade lactic acid bacteria (LAB) as a carrier. The L7/L12 gene was expressed in Lactococcus lactis, the model LAB, under the nisin-inducible promoter. Using different signals, L7/L12 was produced in cytoplasmic, cell-wall-anchored, and secreted forms. Cytoplasmic production of L7/L12 gave a low yield, estimated at 0.5 mg/liter. Interestingly, a secretable form of this normally cytoplasmic protein via fusion with a signal peptide resulted in increased yield of L7/L12 to 3 mg/liter; secretion efficiency (SE) was 35%. A fusion between the mature moiety of the staphylococcal nuclease (Nuc) and L7/L12 further increased yield to 8 mg/liter. Fusion with a synthetic propeptide (LEISSTCDA) previously described as an enhancer for heterologous protein secretion in L. lactis (Y. Le Loir, A. Gruss, S. D. Ehrlich, and P. Langella, J. Bacteriol. 180:1895-1903, 1998) raised the yield to 8 mg/liter and SE to 50%. A surface-anchored L7/L12 form in L. lactis was obtained by fusing the cell wall anchor of Streptococcus pyogenes M6 protein to the C-terminal end of L7/L12. The fusions described allow the production and targeting of L7/L12 in three different locations in L. lactis. This is the first example of a B. abortus antigen produced in a food-grade bacterium and opens new perspectives for alternative vaccine strategies against brucellosis.