Versatile enzyme expression and characterization system for Aspergillus nidulans, with the Penicillium brevicompactum polyketide synthase gene from the mycophenolic acid gene cluster as a test case

Assigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems. To facilitate the use of filamentous fungi in functional genomics, we present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location of Aspergillus nidulans. By a single USER cloning step, genes are easily inserted into a combined targeting-expression cassette ready for rapid integration and analysis. The system comprises a vector set that allows genes to be expressed either from the constitutive PgpdA promoter or from the inducible PalcA promoter. Moreover, by using the vector set, protein variants can easily be made and expressed from the same locus, which is mandatory for proper comparative analyses. Lastly, all individual elements of the vectors can easily be substituted for other similar elements, ensuring the flexibility of the system. We have demonstrated the potential of the system by transferring the 7,745-bp large mpaC gene from Penicillium brevicompactum to A. nidulans. In parallel, we produced defined mutant derivatives of mpaC, and the combined analysis of A. nidulans strains expressing mpaC or mutated mpaC genes unequivocally demonstrated that mpaC indeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid.     

Behaviour of rainbow trout Oncorhynchus mykiss presented with a choice of normoxia and stepwise progressive hypoxia

The objective of this study was to identify behavioural adjustments leading to avoidance of hypoxia. Using the oxygen-sensitive species rainbow trout Oncorhynchus mykiss as a model, individual fish were recorded while moving freely between two sides of a test arena: one with normoxia and one with stepwise progressive hypoxia [80-30% dissolved oxygen (DO) air saturation]. The results demonstrated a gradual decrease in the total time spent in hypoxia starting at 80% DO air saturation. At this DO level, the avoidance of hypoxia could not be attributed to changes in spontaneous swimming speed, neither in normoxia nor in hypoxia. Reducing the DO level to 60% air saturation resulted in decreased spontaneous swimming speed in normoxia, yet the number of trips to the hypoxic side of the test arena remained unchanged. Moreover, data revealed increased average residence time per trip in normoxia at DO levels ≤60% air saturation and decreased average residence time per trip in hypoxia at DO levels ≤50% air saturation. Finally, the spontaneous swimming speed in hypoxia increased at DO levels ≤40% air saturation and the number of trips to hypoxia decreased at the 30% DO air saturation level. Thus, avoidance of the deepest hypoxia was connected with a reduced number of trips to hypoxia as well as decreased and increased spontaneous swimming speed in normoxia and hypoxia, respectively. Collectively, the data support the conclusions that the mechanistic basis for avoidance of hypoxia may (1) not involve changes in swimming speed during mild hypoxia and (2) depend on the severity of hypoxia.     

Pancreatic bicarbonate secretion involves two proton pumps

Pancreas secretes fluid rich in digestive enzymes and bicarbonate. The alkaline secretion is important in buffering of acid chyme entering duodenum and for activation of enzymes. This secretion is formed in pancreatic ducts, and studies to date show that plasma membranes of duct epithelium express H(+)/HCO(3)(-) transporters, which depend on gradients created by the Na(+)/K(+)-ATPase. However, the model cannot fully account for high-bicarbonate concentrations, and other active transporters, i.e. pumps, have not been explored. Here we show that pancreatic ducts express functional gastric and non-gastric H(+)-K(+)-ATPases. We measured intracellular pH and secretion in small ducts isolated from rat pancreas and showed their sensitivity to H(+)-K(+) pump inhibitors and ion substitutions. Gastric and non-gastric H(+)-K(+) pumps were demonstrated on RNA and protein levels, and pumps were localized to the plasma membranes of pancreatic ducts. Quantitative analysis of H(+)/HCO(3)(-) and fluid transport shows that the H(+)-K(+) pumps can contribute to pancreatic secretion in several species. Our results call for revision of the bicarbonate transport physiology in pancreas, and most likely other epithelia. Furthermore, because pancreatic ducts play a central role in several pancreatic diseases, it is of high relevance to understand the role of H(+)-K(+) pumps in pathophysiology.     

Full-length cardiac Na+/Ca2+ exchanger 1 protein is not phosphorylated by protein kinase A

The cardiac Na(+)/Ca(2+) exchanger 1 (NCX1) is an important regulator of intracellular Ca(2+) homeostasis and cardiac function. Several studies have indicated that NCX1 is phosphorylated by the cAMP-dependent protein kinase A (PKA) in vitro, which increases its activity. However, this finding is controversial and no phosphorylation site has so far been identified. Using bioinformatic analysis and peptide arrays, we screened NCX1 for putative PKA phosphorylation sites. Although several NCX1 synthetic peptides were phosphorylated by PKA in vitro, only one PKA site (threonine 731) was identified after mutational analysis. To further examine whether NCX1 protein could be PKA phosphorylated, wild-type and alanine-substituted NCX1-green fluorescent protein (GFP)-fusion proteins expressed in human embryonic kidney (HEK)293 cells were generated. No phosphorylation of full-length or calpain- or caspase-3 digested NCX1-GFP was observed with purified PKA-C and [γ-(32)P]ATP. Immunoblotting experiments with anti-PKA substrate and phosphothreonine-specific antibodies were further performed to investigate phosphorylation of endogenous NCX1. Phospho-NCX1 levels were also not increased after forskolin or isoproterenol treatment in vivo, in isolated neonatal cardiomyocytes, or in total heart homogenate. These data indicate that the novel in vitro PKA phosphorylation site is inaccessible in full-length as well as in calpain- or caspase-3 digested NCX1 protein, suggesting that NCX1 is not a direct target for PKA phosphorylation.     

Inhibitors and promoters of tubulin polymerization: synthesis and biological evaluation of chalcones and related dienones as potential anticancer agents

A series of dihalogenated chalcones and structurally related dienones were synthesized and evaluated for their antiproliferative activity in 10 different cancer cell lines and for their effect on microtubule assembly. All compounds showed cytotoxic activity, with IC(50) values in the 5-280 μM range depending on the chalcone structure and the cell line. Five of the compounds were found to be tubulin polymerization inhibitors. In contrast, one of the compounds was found to stabilize tubulin to the same extent as the anticancer drug docetaxel. Molecular modeling suggested that the tubulin inhibitors bind to the colchicine binding site of β-tubulin while the novel tubulin stabilization agent seems to interact with the paclitaxel binding site.     

Synthesis of glycoconjugate carbonic anhydrase inhibitors by ruthenium-catalysed azide-alkyne 1,3-dipolar cycloaddition

Carbonic anhydrase IX (CA IX) is a recently validated target for the development of new cancer therapies. In this Letter we describe the synthesis and CA inhibition of a novel series of carbohydrate-based 1,5-disubstituted-1,2,3-triazole benzenesulfonamides. The key step of our synthesis is the regioselective Huisgen's 1,3-dipolar cycloaddition reaction (1,3-DCR) from carbohydrate azide substrates and 4-ethynylbenzenesulfonamide using a ruthenium-catalysed azide-alkyne cycloaddition (RuAAC). Our findings identified a number of triazole inhibitors (compounds 18, 19, 21-23, and 26) that block CA IX activity with inhibition constants less than 10 nM. One inhibitor (compound 17) possessed very good selectivity for CA IX over off-target CAs. These CA inhibitors have developmental potential to selectively target cancer cells, leading to cell death.     

Clusters of ligands on dendrimer surfaces

The development of methodology that is designed to allow a significant increase in the patterning and in the functionalization of the dendrimer is the ultimate goal of the research described here. Glycoside clusters based on TRIS were formed using click chemistry and were attached to PAMAM dendrimers. A series of dendrimers bearing tris-mannoside and an ethoxyethanol group was synthesized, and the binding interactions of these dendrimers with Concanavalin A were evaluated using inhibition ELISAs. The results of the inhibition ELISAs suggest that tris-mannoside clusters can replace individual sugars on the dendrimer without loss of function. Since tris-mannoside clustering allows for a redistribution of the dendrimers’ surface functionalities, from this chemistry one can envision patterned dendrimers that incorporate multiple groups to increase the function and utility of the dendrimer.     

Impact of random and systematic recall errors and selection bias in case--control studies on mobile phone use and brain tumors in adolescents (CEFALO study)

Whether the use of mobile phones is a risk factor for brain tumors in adolescents is currently being studied. Case--control studies investigating this possible relationship are prone to recall error and selection bias. We assessed the potential impact of random and systematic recall error and selection bias on odds ratios (ORs) by performing simulations based on real data from an ongoing case--control study of mobile phones and brain tumor risk in children and adolescents (CEFALO study). Simulations were conducted for two mobile phone exposure categories: regular and heavy use. Our choice of levels of recall error was guided by a validation study that compared objective network operator data with the self-reported amount of mobile phone use in CEFALO. In our validation study, cases overestimated their number of calls by 9% on average and controls by 34%. Cases also overestimated their duration of calls by 52% on average and controls by 163%. The participation rates in CEFALO were 83% for cases and 71% for controls. In a variety of scenarios, the combined impact of recall error and selection bias on the estimated ORs was complex. These simulations are useful for the interpretation of previous case-control studies on brain tumor and mobile phone use in adults as well as for the interpretation of future studies on adolescents.