Type 1 inositol-1,4,5-trisphosphate receptor is a late substrate of caspases during apoptosis

Apoptosis is characterized by the proteolytic cleavage of hundreds of proteins. One of them, the type 1 inositol-1,4,5-trisphosphate receptor (IP(3) R-1), a multimeric receptor located on the endoplasmic reticulum (ER) membrane that is critical to calcium homeostasis, was reported to be cleaved during staurosporine (STS) induced-apoptosis in Jurkat cells. Because the reported cleavage site separates the IP(3) binding site from the channel moiety, its cleavage would shut down a critical signaling pathway that is common to several cellular processes. Here we show that IP(3) R-1 is not cleaved in 293 cells treated with STS, TNFα, Trail, or ultra-violet (UV) irradiation. Further, it is not cleaved in Hela or Jurkat cells induced to undergo apoptosis with Trail, TNFα, or UV. In accordance with previous reports, we demonstrate that it is cleaved in a Jurkat cell line treated with STS. However its cleavage occurs only after poly(ADP-ribose) polymerase (PARP), which cleavage is a hallmark of apoptosis, and p23, a poor caspase-7 substrate, are completely cleaved, suggesting that IP(3) R-1 is a relatively late substrate of caspases. Nevertheless, the receptor is fully accessible to proteolysis in cellulo by ectopically overexpressed caspase-7 or by the tobacco etch virus (TEV) protease. Finally, using recombinant caspase-3 and microsomal fractions enriched in IP(3) R-1, we show that the receptor is a poor caspase-3 substrate. Consequently, we conclude that IP(3) R-1 is not a key death substrate.     

Skimming the surface with Burgess Shale arthropod locomotion

The first arthropod trackways are described from the Middle Cambrian Burgess Shale Formation of Canada. Trace fossils, including trackways, provide a rich source of biological and ecological information, including direct evidence of behaviour not commonly available from body fossils alone. The discovery of large arthropod trackways is unique for Burgess Shale-type deposits. Trackway dimensions and the requisite number of limbs are matched with the body plan of a tegopeltid arthropod. Tegopelte, one of the rarest Burgess Shale animals, is over twice the size of all other benthic arthropods known from this locality, and only its sister taxon, Saperion, from the Lower Cambrian Chengjiang biota of China, approaches a similar size. Biomechanical trackway analysis demonstrates that tegopeltids were capable of rapidly skimming across the seafloor and, in conjunction with the identification of gut diverticulae in Tegopelte, supports previous hypotheses on the locomotory capabilities and carnivorous mode of life of such arthropods. The trackways occur in the oldest part (Kicking Horse Shale Member) of the Burgess Shale Formation, which is also known for its scarce assemblage of soft-bodied organisms, and indicate at least intermittent oxygenated bottom waters and low sedimentation rates.     

Association among growth, food consumption-related traits and amylase gene polymorphism in the Pacific oyster Crassostrea gigas

To examine further a previously reported association between amylase gene polymorphism and growth in the Pacific oyster Crassostrea gigas, ecophysiological parameters and biochemical and molecular expression levels of alpha-amylase were studied in Pacific oysters of different amylase genotypes. Genotypes that previously displayed significantly different growth were found to be significantly different for ingestion and absorption efficiency. These estimated parameters, used in a dynamic energy budget model, showed that observed ingestion rates (unlike absorption efficiencies) allowed an accurate prediction of growth potential in these genotypes. The observed association between growth and amylase gene polymorphism is therefore more likely to be related to ingestion than to absorption efficiency. Additionally, relative mRNA levels of the two amylase cDNAs were also strongly associated with amylase gene polymorphism, possibly reflecting variation in an undefined regulatory region, although no corresponding variation was observed in specific amylase activity. Amylase gene sequences were determined for each genotype, showing the existence of only synonymous or functionally equivalent non-synonymous polymorphisms. The observed associations among growth, food consumption-related traits and amylase gene polymorphism are therefore more likely to be related to variation in the level of amylase gene expression than to functional enzymatic variants.     

Controlling Schistosomiasis: Significant Decrease of Anaemia Prevalence One Year after a Single Dose of Praziquantel in Nigerien Schoolchildren

Background

In the framework of the monitoring and evaluation of the Nigerien schistosomiasis and soil-transmitted helminth control programme, a follow-up of children took place in eight sentinel sites. The objective of the study was to assess the evolution of Schistosoma haematobium infection and anaemia in schoolchildren after a single administration of praziquantel (PZQ) and albendazole.

Methods/Principal Findings

Pre-treatment examination and follow-up at one year post-treatment of schoolchildren aged 7, 8, and 11 years, including interview, urine examination, ultrasound examination of the urinary tract, and measurement of haemoglobin. Before treatment, the overall prevalence of S. heamatobium infection was 75.4% of the 1,642 enrolled children, and 21.8% of children excreted more than 50 eggs/10 ml urine. Prevalence increased with age. The overall prevalence of anaemia (haemoglobin <11.5 g/dl) was 61.6%, decreasing significantly with increasing age. The mean haemoglobinemia was 11 g/dl. In bivariate analysis, anaemia was significantly more frequent in children infected with S. haematobium, although it was not correlated to the intensity of infection. Anaemia was also associated with micro-haematuria and to kidney distensions. In a sub-sample of 636 children tested for P. falciparum infection, anaemia was significantly more frequent in malaria-infected children. In multivariate analysis, significant predictors of anaemia were P. falciparum infection, kidney distension, and the village. One year after a single-dose praziquantel treatment (administered using the WHO PZQ dose pole) co-administered with albendazole (400 mg single dose) for de-worming, the prevalence of S. haematobium infection was 38%, while the prevalence of anaemia fell to 50.4%. The mean haemoglobinemia showed a statistically significant increase of 0.39 g/dl to reach 11.4 g/dl. Anaemia was no longer associated with S. haematobium or to P. falciparum infections, or to haematuria or ultrasound abnormalities of the urinary tract.

Conclusions

The high prevalence of anaemia in Nigerien children is clearly a result of many factors and not of schistosomiasis alone. Nevertheless, treatment of schistosomiasis and de-worming were followed by a partial, but significant, reduction of anaemia in schoolchildren, not explainable by any other obvious intervention.     

The Physcomitrium (Physcomitrella) patens PpKAI2L receptors for strigolactones and related compounds function via MAX2-dependent and -independent pathways

In angiosperms, the α/β hydrolase DWARF14 (D14), along with the F-box protein MORE AXILLARY GROWTH2 (MAX2), perceives strigolactones (SL) to regulate developmental processes. The key SL biosynthetic enzyme CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8) is present in the moss Physcomitrium patens, and PpCCD8-derived compounds regulate moss extension. The PpMAX2 homolog is not involved in the SL response, but 13 PpKAI2LIKE (PpKAI2L) genes homologous to the D14 ancestral paralog KARRIKIN INSENSITIVE2 (KAI2) encode candidate SL receptors. In Arabidopsis thaliana, AtKAI2 perceives karrikins and the elusive endogenous KAI2-Ligand (KL). Here, germination assays of the parasitic plant Phelipanche ramosa suggested that PpCCD8-derived compounds are likely noncanonical SLs. (+)-GR24 SL analog is a good mimic for PpCCD8-derived compounds in P. patens, while the effects of its enantiomer (−)-GR24, a KL mimic in angiosperms, are minimal. Interaction and binding assays of seven PpKAI2L proteins pointed to the stereoselectivity toward (−)-GR24 for a single clade of PpKAI2L (eu-KAI2). Enzyme assays highlighted the peculiar behavior of PpKAI2L-H. Phenotypic characterization of Ppkai2l mutants showed that eu-KAI2 genes are not involved in the perception of PpCCD8-derived compounds but act in a PpMAX2-dependent pathway. In contrast, mutations in PpKAI2L-G, and -J genes abolished the response to the (+)-GR24 enantiomer, suggesting that PpKAI2L-G, and -J proteins are receptors for moss SLs.

The study of moss PpKAI2L receptors for strigolactones and related compounds highlights MORE AXILLARY GROWTH2-dependent and -independent pathways for the perception of these compounds.     

Towards calixarene-based prodrugs: Drug release and antibacterial behaviour of a water-soluble nalidixic acid/calix[4]arene ester adduct

A water-soluble calixarene-based heterocyclic podand incorporating a quinolone antibiotic subunit, the nalidixic acid, was synthesised and fully characterised. Its prodrug behaviour was assessed in vitro by HPLC, demonstrating the release of the tethered quinolone in model biological conditions. Microbiological studies performed on various Gram-positive and Gram-negative reference strains showed very interesting antibacterial activities.     

The spirocyclopropyl moiety as a methyl surrogate in the structure of l-fucosidase and l-rhamnosidase inhibitors

Nitrogen-in-the-ring analogues of l-fucose and l-rhamnose were prepared, which feature a spirocyclopropyl moiety in place of the methyl group of the natural sugar. The synthetic route involved a titanium-mediated aminocyclopropanation of a glycononitrile as the key step. Four new spirocyclopropyl iminosugar analogues were generated, which displayed some activity towards l-fucosidase and l-rhamnosidase.     

Spirocyclopropyl pyrrolidines as a new series of alpha-L-fucosidase inhibitors

Polyhydroxy 4-azaspiro[2.4]heptane derivatives (spirocyclopropyl iminosugars) were prepared in four to six steps from readily available protected aldoses. The key step of the reaction sequence involves a titanium-mediated aminocyclopropanation of glycononitriles with subsequent cyclization. Five new polyhydroxypyrrolidines so-obtained have been evaluated for their ability to inhibit 16 glycosidases. One of them exhibits selective inhibition of alpha-L-fucosidase from bovine kidney (Ki=1.6 microM, competitive).     

Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2beta-carboxylic acid methyl ester), a cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min), were synthesized from (R)-cocaine. [(3)H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared from [(3)H]methyl iodide, allowing its in vitro pharmacological evaluation (K(D): 9 nM for DAT and IC(50) > 1000 nM for SERT and NET). Routine production batches of 4.5-9.0 GBq of iv injectable solutions of [(11)C]LBT-999 (with specific radioactivities ranging from 30 to 45 GBq/mumol) were prepared in 25-30 min (HPLC purification and formulation included) using the efficient methylation reagent [(11)C]methyl triflate. The preliminary in vivo pharmacological evaluation of [(11)C]LBT-999, using both biodistributions in rats and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate for quantification with PET of DAT in humans.     

Monitoring Repair of UV-Induced 6-4-Photoproducts with a Purified DDB2 Protein Complex

Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]–[4]. Using a purified DDB2 DRC, we created a probe (“DDB2 proteo-probe”) that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair.