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We have established a protocol allowing transient and inducible coexpression of many foreign genes in Drosophila S2 Schneider cells. With this powerful approach of reverse genetics, we studied the interaction of the protein tyrosine kinases Syk and Lyn with the B cell antigen receptor (BCR). We find that Lyn phosphorylates only the first tyrosine whereas Syk phosphorylates both tyrosines of the BCR immunoreceptor tyrosine-based activation motif (ITAM). Furthermore, we show that Syk is a positive allosteric enzyme, which is strongly activated by the binding to the phosphorylated ITAM tyrosines, thus initiating a positive feedback loop at the receptor. The BCR-dependent Syk activation and signal amplification is efficiently counterbalanced by protein tyrosine phosphatases, the activity of which is regulated by H(2)O(2) and the redox equilibrium inside the cell.

We have studied the apoptotic response of poly(ADP-ribose) polymerase (PARP)-/- cells to different inducers and the consequences of the expression of an uncleavable mutant of PARP on the apoptotic process. The absence of PARP drastically increases the sensitivity of primary bone marrow PARP-/- cells to apoptosis induced by an alkylating agent but not by a topoisomerase I inhibitor CPT-11 or by interleukin-3 removal. cDNA of wild type or of an uncleavable PARP mutant (D214A-PARP) has been introduced into PARP-/- fibroblasts, which were exposed to anti-CD95 or an alkylating agent to induce apoptosis. The expression of D214A-PARP results in a significant delay of cell death upon CD95 stimulation. Morphological analysis shows a retarded cell shrinkage and nuclear condensation. Upon treatment with an alkylating agent, expression of wild-type PARP cDNA into PARP-deficient mouse embryonic fibroblasts results in the restoration of the cell viability, and the D214A-PARP mutant had no further effect on cell recovery. In conclusion, PARP-/- cells are extremely sensitive to apoptosis induced by triggers (like alkylating agents), which activates the base excision repair pathway of DNA, and the cleavage of PARP during apoptosis facilitates cellular disassembly and ensures the completion and irreversibility of the process.

Non-conventional major histocompatibility complex class I molecules are involved in a variety of physiological functions, most at the periphery of the immune system per se. Zinc-alpha(2)-glycoprotein (ZAG), the sole soluble member of this superfamily has been implicated in cachexia, a poorly understood yet life-threatening, severe wasting syndrome. To further ascertain the role of ZAG in lipid metabolism and perhaps the immune system, we inactivated both ZAG alleles by gene targeting in mice. Subjecting these ZAG deficient animals to standard or lipid rich food regimens led to increased body weight in comparison to identically treated wild-type mice. This phenotype appeared to correlate with a significant decrease in adipocytic lipolysis that could not be rescued by several pharmacological agents including beta(3)-adrenoreceptor agonists. Furthermore, in contrast to previously reported data, ZAG was found to be ubiquitously and constitutively expressed, with an especially high level in the mouse liver. No overt immunological phenotype was identified in these animals.

SummaryFactor VIII (FVIII) processing within mammalian cells is demonstrated to be much less efficient than proteins of similar size. The deletion of the B-domain from FVIII improves the level of production, due partly to the increase in mRNA synthesis. We aimed to characterise the cellular fate and the intracellular processing of the FVIII molecule devoid of B-domain. A B-domain deleted factor VIII (BDD-FVIII) possessing a furin consensus cleavage site in the connecting segment between the heavy and the light chain, was produced in CHO cell line. In such cells, FVIII was retained as two single chain products from which a majority was aggregated. The two species were located in Triton X-100 soluble (for 60–80%) and insoluble fractions (for 20–40%). The incubation of the expressing cells with tunicamycin (5 μg/ml) and the treatment of the intracellular species with a mixture of Neuraminidase and N-glycosidase-F revealed that both intracellular species were N-glycosylated. Furin over-expression neither diminished the intracellular FVIII contents nor improved its extracellular production. Intracellular FVIII was degraded through both lysosomal and proteasomal pathways as evidenced by inhibitor treatments (e.g. NH4Cl, leupeptin, clasto-Lactacystin β-lactone and MG-132), pulse-chase analysis and confocal observations. This study demonstrates that a BDD-FVIII expressed in CHO cells is inefficiently processed consecutively to intracellular aggregation, proteasomal degradation, and routage to lysosomes.

Following a vascular injury, factor VIII (FVIII) is rapidly activated by thrombin cleavage at arginine (Arg) 372, 740 and 1689. Activated FVIII, an heterotrimer composed by the association of A1, A2 and A3-C1-C2 domains, is rapidly degraded to limit thrombosis risk. Two main phenomenons that account for the disappearence of FVIIIa consist of an intrinsic dissociation of the trimer due to the loss of A2 domain, and the cleavage of the molecule by activated protein C (APC). APC cleaves FVIIIa at Arg 336 and 562. Mutant FVIII molecules were already generated, with one or two arginines substituted, and a subsequent APC cleavage diminished. Since the thrombotic potential of high plasma levels of FVIII has been described, we aimed to generate new factor VIII molecules where the APC cleavage was only modulated. The ultimate goal being to increase in vivo the FVIIIa half-life. Among the two APC cleavage sites, the sequence around the site 562 was the most conserved between species. This region was therefore chosen to be modified with a prior verification that the amino-acids to be modified were not described in any hemophilic phenotype. Subsequently, the six following mutations were realized in a BDD-FVIII cDNA: Q561N, G563A, N564D, N564A, N564Q and I566M as well as the controls R336I, R562K and R336I+R562K. The constructs were transiently expressed in BHK cells to determine the specific activities of the corresponding molecules. The mutant specific activities, as determined by one- and two-stage clotting assays, ranged from 40 to 94 % of the wild-type except for G563A that was almost inactive. CHO clones expressing FVIII molecules were obtained. The mutants and control FVIII molecules were produced, partially purified on heparin column and further analyzed. The comparative specific activities in a two-stage clotting assay were the following (+/− SD; n=6): BDD-FVIII 100 %, Q561N 105 % +/− 45, G563A 6 % +/− 6, N564D 50 % +/− 23, N564A 45 % +/− 21, N564Q 80 % +/− 26, I566M 100 % +/− 41. The one-stage clotting assay gave identical results than the two-stage assay for each mutant. The mutants were then activated by thrombin (1:1) and the occurrence of FVIII activity was monitored. A 20- to 25-fold increase in FVIII activity was measured within 2 minutes following the addition of thrombin for all mutants. The mutants, except for the inactive G563A, were then analyzed for their resistance to APC by three different assays: an APC resistance kit (Coatest, Chromogenix), an in vitro assay that measured APC sensitivity of FVIIIa and an immunoblot assay that visualized the cleavage efficiency of the A2 fragment. These three assays confirmed the APC resistance of the previously published R336I, R562K and R336I+R562K, as compared with wild-type FVIII. They also revealed that the mutants N564D, N564A and I566M behaved similarly to the wild-type FVIII whereas Q561N and N564Q mutants were partially resistant to APC. The APC resistance ratio were the following 2.3 +/− 0.3 for BDD-FVIII, 2.1 +/− 0.4 for N564Q, 1.7 +/− 0,1 for Q561N, 1.6 +/− 0.3 for R562K and 1.3 +/− 0.3 for R336I+R562K. The N564Q and Q561N mutants exhibited a profile intermediate between wild type FVIII and R562K regarding of the loss of FVIIIa activity that was confirmed on the immunoblot profile. In conclusion we have generated new factor VIII molecules that retained their full procoagulant function while possessing a reduced sensitivity to APC cleavage.