Life-cycle-generation-specific developmental processes are modified in the immediate upright mutant of the brown alga Ectocarpus siliculosus

Development of the sporophyte and gametophyte generations of the brown alga E. siliculosus involves two different patterns of early development, which begin with either a symmetric or an asymmetric division of the initial cell, respectively. A mutant, immediate upright (imm), was isolated that exhibited several characteristics typical of the gametophyte during the early development of the sporophyte generation. Genetic analyses showed that imm is a recessive, single-locus Mendelian factor and analysis of gene expression in this mutant indicated that the regulation of a number of life-cycle-regulated genes is specifically modified in imm mutant sporophytes. Thus, IMM appears to be a regulatory locus that controls part of the sporophyte-specific developmental programme, the mutant exhibiting partial homeotic conversion of the sporophyte into the gametophyte, a phenomenon that has not been described previously.     

Synthesis and biological evaluation of 3-amino-, 3-alkoxy- and 3-aryloxy-6-(hetero)arylpyridazines as potent antitumor agents

Various 3-amino-, 3-aryloxy- and alkoxy-6-arylpyridazines have been synthesized by an electrochemical reductive cross-coupling between 3-amino-, 3-aryloxy- or 3-alkoxy-6-chloropyridazines and aryl or heteroaryl halides. In vitro antiproliferative activity of these products was evaluated against a representative panel of cancer cell lines (HuH7, CaCo-2, MDA-MB-231, HCT116, PC3, NCI-H727, HaCaT) and oncogenicity prevention of the more efficient derivatives was highlighted on human breast cancer cell line MDA-MB 468-Luc prior establishing their interaction with p44/42 and Akt-dependent signaling pathways.     

HIV-1 gp41 envelope residues 650-685 exposed on native virus act as a lectin to bind epithelial cell galactosyl ceramide

The initial step in the interaction between human immunodeficiency virus (HIV-1) and epithelial cells is the binding of HIV-1 envelope glycoproteins to the epithelial cell galactosyl ceramide (GalCer). Here we show that HIV-1 envelope gp41 residues 650-685 bind GalCer in a galactose-specific manner. The gp41 residues that display this lectin activity are highly conserved among HIV-1 isolates and constitute three regions: residues 650-661, which encompass a charged helix; residues 662-667, referred to as the conserved epitope ELDKWA, the epitope recognized by antibodies that neutralize HIV-1 entry in epithelial and CD4(+)-mononucleated cells; and residues 668-685, a hydrophobic Trp-rich sequence that stabilizes the structure of the galactose binding site. Similar to other galactose-specific lectins, the gp41 lectin site is active only as an oligomer. Finally the orientation of the galactose toward the gp41 lectin site appears to be controlled by the lipid microenvironment of the epithelial membrane. From the experimental data we construct a theoretical model of the interaction between gp41 and GalCer based on thermodynamic considerations. This model integrates the dynamics and the spatial organization of the viral envelope glycoproteins, GalCer organized in raft microdomains in the apical region of the epithelial cell membrane and the interfacial water. Characterization of the minimal sequence and structure of gp41 in direct interaction with GalCer may help unravel the still unknown immunogenic determinant able to elicit antibodies against ELDKWA and target of one of the rare neutralizing antibodies against gp41.     

The cytosolic Arabidopsis thaliana cysteine desulfurase ABA3 delivers sulfur to the sulfurtransferase STR18

The biosynthesis of many sulfur-containing molecules depends on cysteine as a sulfur source. Both the cysteine desulfurase (CD) and rhodanese (Rhd) domain–containing protein families participate in the trafficking of sulfur for various metabolic pathways in bacteria and human, but their connection is not yet described in plants. The existence of natural chimeric proteins containing both CD and Rhd domains in specific bacterial genera, however, suggests a general interaction between these proteins. We report here the biochemical relationships between two cytosolic proteins from Arabidopsis thaliana, a Rhd domain–containing protein, the sulfurtransferase 18 (STR18), and a CD isoform referred to as ABA3, and compare these biochemical features to those of a natural CD–Rhd fusion protein from the bacterium Pseudorhodoferax sp. We observed that the bacterial enzyme is bifunctional exhibiting both CD and STR activities using l-cysteine and thiosulfate as sulfur donors but preferentially using l-cysteine to catalyze transpersulfidation reactions. In vitro activity assays and mass spectrometry analyses revealed that STR18 stimulates the CD activity of ABA3 by reducing the intermediate persulfide on its catalytic cysteine, thereby accelerating the overall transfer reaction. We also show that both proteins interact in planta and form an efficient sulfur relay system, whereby STR18 catalyzes transpersulfidation reactions from ABA3 to the model acceptor protein roGFP2. In conclusion, the ABA3–STR18 couple likely represents an uncharacterized pathway of sulfur trafficking in the cytosol of plant cells, independent of ABA3 function in molybdenum cofactor maturation.     

Production of Mucosally Transmissible SHIV Challenge Stocks from HIV-1 Circulating Recombinant Form 01_AE env Sequences

Simian-human immunodeficiency virus (SHIV) challenge stocks are critical for preclinical testing of vaccines, antibodies, and other interventions aimed to prevent HIV-1. A major unmet need for the field has been the lack of a SHIV challenge stock expressing circulating recombinant form 01_AE (CRF01_AE) env sequences. We therefore sought to develop mucosally transmissible SHIV challenge stocks containing HIV-1 CRF01_AE env derived from acutely HIV-1 infected individuals from Thailand. SHIV-AE6, SHIV-AE6RM, and SHIV-AE16 contained env sequences that were >99% identical to the original HIV-1 isolate and did not require in vivo passaging. These viruses exhibited CCR5 tropism and displayed a tier 2 neutralization phenotype. These challenge stocks efficiently infected rhesus monkeys by the intrarectal route, replicated to high levels during acute infection, and established chronic viremia in a subset of animals. SHIV-AE16 was titrated for use in single, high dose as well as repetitive, low dose intrarectal challenge studies. These SHIV challenge stocks should facilitate the preclinical evaluation of vaccines, monoclonal antibodies, and other interventions targeted at preventing HIV-1 CRF01_AE infection.     

Prolonged elevated levels of c-kit+ progenitor cells after a myocardial infarction by beta 2 adrenergic receptor priming

Endogenous progenitor cells may participate in cardiac repair after a myocardial infarction (MI). The beta 2 adrenergic receptor (ß2-AR) pathway induces proliferation of c-kit+ cardiac progenitor cells (CPC) in vitro. We investigated if ß2-AR pharmacological stimulation could ameliorate endogenous CPC-mediated regeneration after a MI. C-kit+ CPC ß1-AR and ß2-AR expression was evaluated in vivo and in vitro. A significant increase in the percentage of CPCs expressing ß1-AR and ß2-AR was measured 7 days post-MI. Accordingly, 24 hrs of low serum and hypoxia in vitro significantly increased CPC ß2-AR expression. Cell viability and differentiation assays validated a functional role of CPC ß2-AR. The effect of pharmacological activation of ß2-AR was studied in C57 mice using fenoterol administered in the drinking water 1 week before MI or sham surgery or at the time of the surgery. MI induced a significant increase in the percentage of c-kit+ progenitor cells at 7 days, whereas pretreatment with fenoterol prolonged this response resulting in a significant elevated number of CPC up to 21 days post-MI. This increased number of CPC correlated with a decrease in infarct size. The immunofluorescence analysis of the heart tissue for proliferation, apoptosis, macrophage infiltration, cardiomyocytes surface area, and vessel density showed significant changes on the basis of surgery but no benefit due to fenoterol treatment. Cardiac function was not ameliorated by fenoterol administration when evaluated by echocardiography. Our results suggest that ß2-AR stimulation may improve the cardiac repair process by supporting an endogenous progenitor cell response but is not sufficient to improve the cardiac function.     

Physical mapping and cloning of RAD56

Here we report the physical mapping of the rad56-1 mutation to the NAT3 gene, which encodes the catalytic subunit of the NatB N-terminal acetyltransferase in Saccharomyces cerevisiae. Mutation of RAD56 causes sensitivity to X-rays, methyl methanesulfonate, zeocin, camptothecin and hydroxyurea, but not to UV light, suggesting that N-terminal acetylation of specific DNA repair proteins is important for efficient DNA repair.     

Snake venoms as a source of compounds modulating sperm physiology: Secreted phospholipases A2 from Oxyuranus scutellatus scutellatus impact sperm motility,acrosome reaction and in vitro fertilization in mice

The goal of this study was to identify new compounds from venoms able to modulate sperm physiology and more particularly sperm motility. For this purpose, we screened the effects of 16 snake venoms cleared of molecules higher than 15 kDa on sperm motility. Venoms rich in neurotoxins like those from Oxyuranus scutellatus scutellatus or Daboia russelii, were highly potent inhibitors of sperm motility. In contrast, venoms rich in myotoxins like those from Echis carinatus, Bothrops alternatus and Macrovipera lebetina, were inactive. From the main pharmacologically-active fraction of the Taipan snake O. scutellatus s., a proteomic approach allowed us to identify 16 different proteins, among which OS1 and OS2, two secreted phospholipases A2 (sPLA₂). Purified OS1 and OS2 mimicked the inhibitory effect on sperm motility and were likely responsible for the inhibitory effect of the active fraction. OS1 and OS2 triggered sperm acrosome reaction and induced lipid rearrangements of the plasma membrane. The catalytic activity of OS2 was required to modulate sperm physiology since catalytically inactive mutants had no effect. Finally, sperm treated with OS2 were less competent than control sperm to initiate in vitro normal embryo development. This is the first report characterizing sPLA₂ toxins that modulate in vitro sperm physiology.