CUL4 associates with DDB1 and DET1 and its downregulation affects diverse aspects of development in Arabidopsis thaliana

Cullins are central scaffolding subunits in eukaryotic E3 ligases that facilitate the ubiquitination of target proteins. Arabidopsis contains at least 11 cullin proteins but only a few of them have been assigned biological roles. In this work Arabidopsis cullin 4 is shown to assemble with DDB1, RBX1, DET1 and DDB2 in vitro and in planta. In addition, by using T-DNA insertion and CUL4 antisense lines we demonstrate that corresponding mutants are severely affected in different aspects of development. Reduced CUL4 expression leads to a reduced number of lateral roots, and to abnormal vascular tissue and stomatal development. Furthermore, cul4 mutants display a weak constitutive photomorphogenic phenotype. These results therefore assign an important function to CUL4 during plant development and provide strong evidence that CUL4 assembles together with RBX1 and DDB1 proteins to form a functional E3 ligase in Arabidopsis.     

Diaryl substituted pyrrolidinones and pyrrolones as 5-HT2C inhibitors: synthesis and biological evaluation

Within the continuous quest for the discovery of novel compounds able to treat anxiety and depression, the generation of a pharmacophore model for 5-HT2C receptor antagonists and the discovery of a new class of potent and selective 5-HT2C molecules are reported.     

LANCL2 Is Necessary for Abscisic Acid Binding and Signaling in Human Granulocytes and in Rat Insulinoma Cells

Abscisic acid (ABA) is a plant hormone regulating fundamental physiological functions in plants, such as response to abiotic stress. Recently, ABA was shown to be produced and released by human granulocytes, by insulin-producing rat insulinoma cells, and by human and murine pancreatic β cells. ABA autocrinally stimulates the functional activities specific for each cell type through a receptor-operated signal transduction pathway, sequentially involving a pertussis toxin-sensitive receptor/G-protein complex, cAMP, CD38-produced cADP-ribose and intracellular calcium. Here we show that the lanthionine synthetase C-like protein LANCL2 is required for ABA binding on the membrane of human granulocytes and that LANCL2 is necessary for transduction of the ABA signal into the cell-specific functional responses in granulocytes and in rat insulinoma cells. Co-expression of LANCL2 and CD38 in the human HeLa cell line reproduces the ABA-signaling pathway. Results obtained with granulocytes and CD38⁺/LANCL2⁺ HeLa transfected with a chimeric G-protein (Gα_q/i) suggest that the pertussis toxin-sensitive G-protein coupled to LANCL2 is a G_i. Identification of LANCL2 as a critical component of the ABA-sensing protein complex will enable the screening of synthetic ABA antagonists as prospective new anti-inflammatory and anti-diabetic agents.The plant hormone abscisic acid (ABA)⁴ plays a fundamental role in the regulation of plant response to environmental conditions, as well as in plant tissue development (1). Although the ABA biosynthetic pathway in plants and in fungi has been largely detailed, identification of the components of the ABA signaling pathway, particularly of the ABA receptor(s), has remained elusive. Two ABA-binding proteins have been identified in different plant tissues: the chloroplast Mg-chelatase subunit H (2) and, most recently, the G-protein-coupled receptor GCR2, which appears to mediate ABA-controlled stomatal closure and seed dormancy in Arabidopsis (3), although the role of GCR2 in the control of seed germination is still controversial (4–6) and its coupling to a G-protein has been refuted on the basis of sequence analyses (7–8). The Mg-chelatase subunit H was proposed as an intracellular ABA receptor, whereas GCR2 is a plasmamembrane protein, which interacts with the only Gα subunit (GPA 1) present in Arabidopsis (3). Although the Mg-chelatase subunit H does not show any significant homology with mammalian proteins, GCR2 shares a high amino acid identity with the mammalian peptide-modifying lanthionine synthetase C-like protein (LANCL) family (7). The animal LANCL protein family in turn shows structural similarities with the prokaryotic lanthionine synthetase component C proteins (9) involved in the synthesis of lanthionine-containing antimicrobial peptides known as lantibiotics (10).The fact that lantibiotics are not produced in animals suggests that LANCL proteins have a different function than prokaryotic lanthionine synthetase component C proteins. The human genome contains three LANCL genes, LANCL1, LANCL2, and LANCL3, located on chromosomes 2 and 7 and the X chromosome, respectively (11, 12). LANCL1 was the first member of the family to be isolated from human erythrocyte membranes (13). The LANCL2 mRNA was identified in a screening procedure for genes whose down-regulation resulted in anticancer drug resistance; thus, LANCL2 was also called testis-specific Adriamicin sensitivity protein (14). The structural assignment for the human LANCL proteins remains controversial. Based on the presence of seven putative transmembrane domains, LANCL1 and -2 were originally described as new G-protein-coupled receptors (GPCR69A and GPR69B, respectively); however, subsequent studies performed on human epithelial cells overexpressing LANCL1 or LANCL2 fused to the green fluorescent protein (LANCL1-GFP and LANCL2-GFP) showed that LANCL1-GFP is mainly found in the cytosol and in the nucleus, whereas LANCL2-GFP is associated with the plasmamembrane through N-terminal myristoylation (15). Similarly, the debate over the structurally related GCR2 is still open (3–6, 8).ABA has recently been demonstrated to be an endogenous pro-inflammatory hormone in human granulocytes, stimulating several cell functions (phagocytosis, reactive oxygen species and nitric oxide production, chemotaxis, and chemokinesis) through a pathway involving a pertussis toxin (PTX)-sensitive G-protein/receptor complex located on the plasmamembrane, cAMP overproduction, protein kinase A-dependent phosphorylation of the human ADP-ribosyl cyclase CD38, and consequent cADP-ribose (cADPR) generation, leading to an increase of the intracellular Ca²⁺ concentration (16; see also Ref. 17). This signaling pathway is similar to that triggered by ABA in plants (18). Fluorescence microscopy confirmed binding of biotinylated ABA to the granulocyte plasmamembrane. Scatchard plot analysis of [³H]ABA binding demonstrated presence of both high and low affinity ABA binding sites (K_d 11 nm and 500 μm, respectively) on human granulocytes (16). Most recently, nanomolar ABA has been shown to stimulate insulin secretion by human and murine pancreatic β cells and by rat insulinoma cell lines through a signaling pathway similar to the one described in human granulocytes (19). The autocrine release of ABA from glucose-stimulated human and rodent insulin-releasing cells, together with the fact that ABA is also produced by activated inflammatory cells, granulocytes (16), and monocytes (20), suggests that this hormone may contribute to the network of cytokine signals exchanged between inflammatory cells and pancreatic β cells, which is increasingly recognized as a fundamental mechanism in the development of the metabolic syndrome and type II diabetes (21–24).Based on (i) the sequence homology between the putative Arabidopsis ABA-receptor protein GCR2 and the human LANCL protein family, and (ii) the reported association of LANCL2 with the plasmamembrane, we investigated whether LANCL2 might be involved in ABA sensing in mammalian ABA-responsive cells. The results obtained indicate that LANCL2 is indeed, (i) required for ABA binding to the plasmamembrane of human granulocytes and (ii) necessary for the activation of the ABA signaling pathway, leading to the stimulation of the functional responses induced by ABA in human granulocytes and in rat insulinoma cells.     

A dynamical feedback model for adaptation in the olfactory transduction pathway

Olfactory transduction exhibits two distinct types of adaptation, which we denote multipulse and step adaptation. In terms of measured transduction current, multipulse adaptation appears as a decrease in the amplitude of the second of two consecutive responses when the olfactory neuron is stimulated with two brief pulses. Step adaptation occurs in response to a sustained steplike stimulation and is characterized by a return to a steady-state current amplitude close to the prestimulus value, after a transient peak. In this article, we formulate a dynamical model of the olfactory transduction pathway, which includes the kinetics of the CNG channels, the concentration of Ca ions flowing through them, and the Ca-complexes responsible for the regulation. Based on this model, a common dynamical explanation for the two types of adaptation is suggested. We show that both forms of adaptation can be well described using different time constants for the kinetics of Ca ions (faster) and the kinetics of the feedback mechanisms (slower). The model is validated on experimental data collected in voltage-clamp conditions using different techniques and animal species.     

Mutations in MAP3K1 cause 46,XY disorders of sex development and implicate a common signal transduction pathway in human testis determination

Investigations of humans with disorders of sex development (DSDs) resulted in the discovery of many of the now-known mammalian sex-determining genes, including SRY, RSPO1, SOX9, NR5A1, WT1, NR0B1, and WNT4. Here, the locus for an autosomal sex-determining gene was mapped via linkage analysis in two families with 46,XY DSD to the long arm of chromosome 5 with a combined, multipoint parametric LOD score of 6.21. A splice-acceptor mutation (c.634-8T>A) in MAP3K1 segregated with the phenotype in the first family and disrupted RNA splicing. Mutations were demonstrated in the second family (p.Gly616Arg) and in two of 11 sporadic cases (p.Leu189Pro, p.Leu189Arg)-18% prevalence in this cohort of sporadic cases. In cultured primary lymphoblastoid cells from family 1 and the two sporadic cases, these mutations altered the phosphorylation of the downstream targets, p38 and ERK1/2, and enhanced binding of RHOA to the MAP3K1 complex. Map3k1 within the syntenic region was expressed in the embryonic mouse gonad prior to, and after, sex determination. Thus, mutations in MAP3K1 that result in 46,XY DSD with partial or complete gonadal dysgenesis implicate this pathway in normal human sex determination.     

A novel small-molecule inhibitor of IL-6 signalling

A small library of pyrrolidinesulphonylaryl molecules has been synthesized via an efficient 4-step route, and members evaluated for their ability to inhibit IL-6 signalling. One molecule (6a) was found to have promising activity against IL-6/STAT3 signalling at the low micromolar level, and to selectively inhibit phosphorylation of STAT3 (but not STAT1) in IL-6 stimulated MDA-MB-231 breast cancer and HeLa cell lines. It was also selectively cytostatic in MDA-MB-231 (STAT3-dependent) versus A4 (STAT3-null) cells suggesting STAT3-specific inhibitory properties.     

Synthesis and biological evaluation of a novel series of bis-salicylaldehydes as mushroom tyrosinase inhibitors

A series of novel bis-salicylaldehydes were synthesised and evaluated as tyrosinase inhibitors using a tyrosinase-dependent l-DOPA oxidation assay. The bis-salicylaldehydes exhibited greater inhibitory activity than salicylaldehyde. Our data suggests that these novel compounds may serve as a structural template for the design and development of novel tyrosinase inhibitors.     

Candida zemplinina can reduce acetic acid produced by Saccharomyces cerevisiae in sweet wine fermentations

In this study we investigated the possibility of using Candida zemplinina, as a partner of Saccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-five C. zemplinina strains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations with S. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels of C. zemplinina started to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pure S. cerevisiae fermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations of S. cerevisiae and C. zemplinina resulted in a decrease of ~0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixed S. cerevisiae and C. zemplinina fermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to the S. cerevisiae osmotic stress response.     

A novel hyperthermostable 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus

We report herein the first molecular characterization of 5'-deoxy-5'-methylthio-adenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII). The isolated gene of SsMTAPII was overexpressed in Escherichia coli BL21. Purified recombinant SsMTAPII is a homohexamer of 180 kDa with an extremely low Km (0.7 microm) for 5'-deoxy-5'-methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120 degrees C and extremely thermostable with an apparent Tm of 112 degrees C that increases in the presence of substrates. The enzyme is characterized by high kinetic stability and remarkable SDS resistance and is also resistant to guanidinium chloride-induced unfolding with a transition midpoint of 3.3 m after 22-h incubation. Limited proteolysis experiments indicated that the only one proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. Moreover, the binding of 5'-deoxy-5'-methylthioadenosine induces a conformational transition that protected the enzyme against protease inactivation. By site-directed mutagenesis we demonstrated that Cys259, Cys261 and Cys262 play an important role in the enzyme stability since the mutants C259S/C261S and C262S show thermophilicity and thermostability features significantly lower than those of the wild-type enzyme. In order to get insight into the physiological role of SsMTAPII a comparative kinetic analysis with the homologous 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was carried out. Finally, the alignment of the protein sequence of SsMTAPII with those of SsMTAP and human 5'-deoxy-5'-methylthioadenosine phosphorylase (hMTAP) shows several key residue changes that may account why SsMTAPII, unlike hMTAP, is able to recognize adenosine as substrate.