Profiling of Androgen Response in Rainbow Trout Pubertal Testis: Relevance to Male Gonad Development and Spermatogenesis

The capacity of testicular somatic cells to promote and sustain germ cell differentiation is largely regulated by sexual steroids and notably androgens. In fish species the importance of androgens is emphasized by their ability to induce sex reversal of the developing fries and to trigger spermatogenesis. Here we studied the influence of androgens on testicular gene expression in trout testis using microarrays. Following treatment of immature males with physiological doses of testosterone or 11-ketotestosterone, 418 genes that exhibit changes in expression were identified. Interestingly, the activity of testosterone appeared stronger than that of 11-ketotestosterone. Expression profiles of responsive genes throughout testis development and in isolated germ cells confirmed androgens to mainly affect gene expression in somatic cells. Furthermore, specific clusters of genes that exhibit regulation coincidently with changes in the natural circulating levels of androgens during the reproductive cycle were highlighted, reinforcing the physiological significance of these data. Among somatic genes, a phylogenetic footprinting study identified putative androgen response elements within the proximal promoter regions of 42 potential direct androgen target genes. Finally, androgens were also found to alter the germ line towards meiotic expression profiles, supporting the hypothesis of a role for the somatic responsive genes in driving germ cell fate. This study significantly increases our understanding of molecular pathways regulated by androgens in vertebrates. The highly cyclic testicular development in trout together with functions associated with regulated genes reveal potential mechanisms for androgen actions in tubule formation, steroid production, germ cell development and sperm secretion.     

RefZ Facilitates the Switch from Medial to Polar Division during Spore Formation in Bacillus subtilis

During sporulation, Bacillus subtilis redeploys the division protein FtsZ from midcell to the cell poles, ultimately generating an asymmetric septum. Here, we describe a sporulation-induced protein, RefZ, that facilitates the switch from a medial to a polar FtsZ ring placement. The artificial expression of RefZ during vegetative growth converts FtsZ rings into FtsZ spirals, arcs, and foci, leading to filamentation and lysis. Mutations in FtsZ specifically suppress RefZ-dependent division inhibition, suggesting that RefZ may target FtsZ. During sporulation, cells lacking RefZ are delayed in polar FtsZ ring formation, spending more time in the medial and transition stages of FtsZ ring assembly. A RefZ-green fluorescent protein (GFP) fusion localizes in weak polar foci at the onset of sporulation and as a brighter midcell focus at the time of polar division. RefZ has a TetR DNA binding motif, and point mutations in the putative recognition helix disrupt focus formation and abrogate cell division inhibition. Finally, chromatin immunoprecipitation assays identified sites of RefZ enrichment in the origin region and near the terminus. Collectively, these data support a model in which RefZ helps promote the switch from medial to polar division and is guided by the organization of the chromosome. Models in which RefZ acts as an activator of FtsZ ring assembly near the cell poles or as an inhibitor of the transient medial ring at midcell are discussed.     

The dynamics of replication licensing in live Caenorhabditis elegans embryos

Accurate DNA replication requires proper regulation of replication licensing, which entails loading MCM-2-7 onto replication origins. In this paper, we provide the first comprehensive view of replication licensing in vivo, using video microscopy of Caenorhabditis elegans embryos. As expected, MCM-2-7 loading in late M phase depended on the prereplicative complex (pre-RC) proteins: origin recognition complex (ORC), CDC-6, and CDT-1. However, many features we observed have not been described before: GFP-ORC-1 bound chromatin independently of ORC-2-5, and CDC-6 bound chromatin independently of ORC, whereas CDT-1 and MCM-2-7 DNA binding was interdependent. MCM-3 chromatin loading was irreversible, but CDC-6 and ORC turned over rapidly, consistent with ORC/CDC-6 loading multiple MCM-2-7 complexes. MCM-2-7 chromatin loading further reduced ORC and CDC-6 DNA binding. This dynamic behavior creates a feedback loop allowing ORC/CDC-6 to repeatedly load MCM-2-7 and distribute licensed origins along chromosomal DNA. During S phase, ORC and CDC-6 were excluded from nuclei, and DNA was overreplicated in export-defective cells. Thus, nucleocytoplasmic compartmentalization of licensing factors ensures that DNA replication occurs only once.     

Discovery of highly potent Src SH2 binders: structure-activity studies and X-ray structures

Optimization of the hydrophobic moiety of caprolactam/thiazepinone based compounds led to the identification of potent Src SH2 binders in two different series incorporating a phosphotyrosine group (RU 81843) or a phosphobenzoic group (RU 79181). The X-ray co-structures with the Src SH2 domain revealed different binding modes for RU 81843 and RU 79181, and an excellent fit between RU81843 and the Src SH2 protein thus explaining its high potency (9 nM, 15-fold more potent than pYEEI reference peptide).     

Functional Characterization of Autoantibodies against Complement Component C3 in Patients with Lupus Nephritis

Lupus nephritis (LN) is a complication of the autoimmune disease systemic lupus erythematosus. Because the complement system plays a critical role in orchestrating inflammatory and immune responses as well as in the clearance of immune complexes, autoreactivity to complement components may have considerable pathological consequences. Autoantibodies against the central complement component C3 have been reported in systemic lupus erythematosus, but their molecular mechanism and functional relevance are not well understood. The objective of this study was to evaluate the frequency and the functional properties of the anti-C3 autoantibodies. Anti-C3 autoantibodies were measured in plasma of 39 LN patients, and identification of their epitopes on the C3 molecule was performed. By using surface plasmon resonance, we analyzed the influence of patient-derived IgG antibodies on the interaction of C3b with Factor B, Factor H, and complement receptor 1. The capacity of these antibodies to dysregulate the C3 convertase on the surface of endothelial cell was measured by flow cytometry. Here we report that the frequency of anti-C3 autoantibodies in LN is ∼30%. They inhibited interactions of the negative complement regulators Factor H and complement receptor 1 with C3b. An enhanced C3 deposition was also observed on human endothelial cells in the presence of C3 autoantibodies. In addition, anti-C3 autoantibody levels correlated with disease activity. In conclusion, the anti-C3 autoantibodies in LN may contribute to the autoimmune pathology by their capacity to overactivate the complement system.     

Bothrops lanceolatus snake venom impairs mitochondrial respiration and induces DNA release in human heart preparation

IntroductionEnvenomations by Bothrops snakebites can induce overwhelming systemic inflammation ultimately leading to multiple organ system failure and death. Release of damage-associated molecular pattern molecules (DAMPs), in particular of mitochondrial origin, has been implicated in the pathophysiology of the deregulated innate immune response.ObjectiveTo test whether whole Bothrops lanceolatus venom would induce mitochondrial dysfunction and DAMPs release in human heart preparations.MethodsHuman atrial trabeculae were obtained during cannulation for cardiopulmonary bypass from patients who were undergoing routine coronary artery bypass surgery. Cardiac fibers were incubated with vehicle and whole Bothrops lanceolatus venom for 24hr before high-resolution respirometry, mitochondrial membrane permeability evaluation and quantification of mitochondrial DNA.ResultsCompared with vehicle, incubation of human cardiac muscle with whole Bothrops lanceolatus venom for 24hr impaired respiratory control ratio and mitochondrial membrane permeability. Levels of mitochondrial DNA increased in the medium of cardiac cell preparation incubated with venom of Bothrops lanceolatus.ConclusionOur study suggests that whole venom of Bothrops lanceolatus impairs mitochondrial oxidative phosphorylation capacity and increases mitochondrial membrane permeability. Cardiac mitochondrial dysfunction associated with mitochondrial DAMPs release may alter myocardium function and engage the innate immune response, which may both participate to the cardiotoxicity occurring in patients with severe envenomation.     

A novel type-1 ribosome-inactivating protein isolated from the supernatant of transformed suspension cultures of Trichosanthes kirilowii

A type-1 ribosome-inactivating protein (RIP) designated TK-35 has been purified from the supernatant of suspension cultures of Agrobacterium rhizogenes-transformed stem sections of Trichosanthes kirilowii. The protein was purified from the supernatant by PerSeptive SH/M cation exchange and Sephadex G-75 S gel permeation chromatography. The protein occurs as a monomer, with a molecular weight of 35,117, and is glycosylated. A protein translation inhibition assay indicates that TK-35 has an IC₅₀ value of 2.45 nm and is able to release the rRNA N-glycosidase diagnostic fragment from rabbit reticulocyte lysate. TK-35 is quite thermally stable. Analysis of its N-terminal sequence and two lys-C-protease-digested polypeptides (internal) amino acid sequence indicates that this protein is not homologous to trichosanthin and other type-1 RIPs in Cucurbitaceae family. Received: 20 August 1997 / Revision received: 30 September 1997 / Accepted: 11 November 1997     

Biosynthesis of cell wall mannan in the conidium and the mycelium of Aspergillus fumigatus

The galactomannan is a major cell wall molecule of Aspergillus fumigatus. This molecule is composed of a linear mannan with a repeating unit composed of four α1,6 and α1,2 linked mannose with side chains of galactofuran. To obtain a better understanding of the mannan biosynthesis in A. fumigatus, it was decided to undertake the successive deletion of the 11 genes which are putative orthologs of the mannosyltransferases responsible for establishing α1,6 and α1,2 mannose linkages in yeast. These deletions did not lead to a reduction of the mannan content of the cell wall of the mycelium of A. fumigatus. In contrast, the mannan content of the conidial cell wall was reduced and this reduction was associated with a partial disorganization of the cell wall leading to defects in conidial survival both in vitro and in vivo.