Design and synthesis of a peptide derived from positions 195–244 of human cdc25C phosphatase

We have designed, synthesized and purified a 51 amino acid peptide derived from an essential domain of human cdc25C phosphatase. In vivo, differential phosphorylation of this domain regulates either the induction of mitotic processes, or the checkpoint arrest of eukaryotic cells in response to DNA damage. Peptide synthesis was achieved using the stepwise Fmoc strategy and resulted in an important yield of highly pure peptide. The final peptide was identified by amino acid analysis, electrospray mass spectrometry and nuclear magnetic resonance, which revealed that one of the two methionines within the peptide was oxidized into its sulphoxide derivative We investigated whether this 51 amino acid peptide folded into secondary structures in solution by circular dichroism and observed the formation of alpha helices in TFE. Finally, we verified that this peptide could bind to its biologically relevant 14‐3‐3 partner in vitro by fluorescence spectroscopy. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Semaphorin 3F and Neuropilin-2 Control the Migration of Human T-Cell Precursors

Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions.     

Anopheles and Plasmodium: from laboratory models to natural systems in the field

Parasites that cause malaria must complete a complex life cycle in Anopheles vector mosquitoes in order to be transmitted from human to human. Previous gene-silencing studies have shown the influence of mosquito immunity in controlling the development of Plasmodium. Thus, parasite survival to the oocyst stage increased when the parasite antagonist gene LRIM1 (leucine-rich repeat immune protein 1) of the mosquito was silenced, but decreased when the C-type lectin agonist gene CTL4 or CTLMA2 (CTL mannose binding 2) was silenced. However, such effects were shown for infections of the human mosquito vector Anopheles gambiae with the rodent parasite Plasmodium berghei. Here, we report the first results of A. gambiae gene silencing on infection by sympatric field isolates of the principal human pathogen P. falciparum. In contrast with the results obtained with the rodent parasite, silencing of the same three genes had no effect on human parasite development. These results highlight the importance of following up discoveries in laboratory model systems with studies on natural parasite-mosquito interactions.     

Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus

We previously documented persistent regulation of erythropoietin (Epo) secretion in mice after a single intramuscular (i.m.) injection of a recombinant adeno-associated virus (rAAV) vector harboring both the tetracycline-dependent transactivator (rtTA) and the Epo cDNA (D. Bohl, A. Salvetti, P. Moullier, and J. M. Heard, Blood 92:1512-1517, 1998). Using the same vector harboring the cynomolgus macaque Epo cDNA instead, the present study evaluated the ability of the tetracycline-regulatable (tetR) system to establish long-term transgene regulation in nonhuman primates. The vector was administered i.m., after which 5-day induction pulses were performed monthly for up to 13 months by using doxycycline (DOX), a tetracycline analog. We show that initial inductions were successful in all individuals and that there was a tight regulation and a rapid deinduction pattern upon DOX withdrawal. For one macaque, regulation of Epo secretion was maintained during the entire experimental period; for the five remaining macaques, secreted Epo became indistinguishable from endogenous Epo upon repeated DOX inductions. We investigated the mechanism involved and showed that, except in the animal in which secretion persisted, delayed humoral and cellular immune responses were directed against the rtTA transactivator protein associated with the reduction of vector DNA in transduced muscles. This study provides some evidence that, when the immune system is not mobilized against the rtTA transactivator, the tetR-regulatable system is able to support long-term transgene regulation in the context of an rAAV in nonhuman primates. In addition, our results suggest potential improvements for vector design.     

Structural Properties of HIV Integrase��Lens Epithelium-derived Growth Factor Oligomers

Integrase (IN) is the catalytic component of the preintegration complex, a large nucleoprotein assembly critical for the integration of the retroviral genome into a host chromosome. Although partial crystal structures of human immunodeficiency virus IN alone and its complex with the integrase binding domain of the host factor PSIP1/lens epithelium-derived growth factor (LEDGF)/p75 are available, many questions remain regarding the properties and structures of LEDGF-bound IN oligomers. Using analytical ultracentrifugation, multiangle light scattering, and small angle x-ray scattering, we have established the oligomeric state, stoichiometry, and molecular shapes of IN·LEDGF complexes in solution. Analyses of intact IN tetramers bound to two different LEDGF truncations allow for placement of the integrase binding domain by difference analysis. Modeling of the small angle x-ray scattering envelopes using existing structural data suggests domain arrangements in the IN oligomers that support and extend existing biochemical data for IN·LEDGF complexes and lend new insights into the quaternary structure of LEDGF-bound IN tetramers. These IN oligomers may be involved in stages of the viral life cycle other than integration, including assembly, budding, and early replication.     

The different steps of skin formation in vertebrates

Skin morphogenesis occurs following a continuous series of cell-cell interactions which can be subdivided into three main stages: 1- the formation of a dense dermis and its overlying epidermis in the future appendage fields (macropattern); 2- the organization of these primary homogeneous fields into heterogeneous ones by the appearance of cutaneous appendage primordia (micropattern) and 3- cutaneous appendage organogenesis itself. In this review, we will first show, by synthesizing novel and previously published data from our laboratory, how heterogenetic and heterospecific dermal/epidermal recombinations have allowed us to distinguish between the respective roles of the dermis and the epidermis. We will then summarize what is known from the work of many different research groups about the molecular signaling which mediates these interactions in order to introduce the following articles of this Special Issue and to highlight what remains to done.     

Pharmacomodulation of a sulfamide 5-HT6 receptor ligand

A series of N-omega-aminoalkyl- or N-omega-amidinoalkyl-2,4,6-triisopropyl benzenesulfonamides has been synthesized and their respective affinity indices on 5-HT6 receptor determined. This evaluation clearly showed that the compounds possessing an arylpiperazine moiety or an amidine function exhibited good affinity for the model.