Diphenylcyclohexylamine derivatives as new potent multidrug resistance (MDR) modulators

A series of compounds with a diphenylmethyl cyclohexyl skeleton, loosely related to verapamil, has been synthesized and tested as MDR modulators on anthracycline-resistant erythroleukemia K 562 cells. Their residual cardiovascular action (negative inotropic and chronotropic activity as well as vasorelaxant activity) was evaluated on guinea-pig isolated atria preparations and on guinea-pig aortic strip preparations. Most compounds of the series possess a good MDR-reverting activity together with a low cardiovascular action. Among them, compounds 3a1, 7a, and 8a are more potent than verapamil as MDR reverters and lack any cardiovascular action; they can represent useful leads for the development of new safe MDR reversing drugs.     

Nicking activity on pBR322 DNA of ribosome inactivating proteins from Phytolacca dioica L. leaves

Ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves are rRNA-N-glycosidases, as well as adenine polynucleotide glycosylases. Here we report that some of them cleave supercoiled pBR322 dsDNA, generating relaxed and linear molecules. PD-L1, the glycosylated major form isolated from the winter leaves of adult P . dioica plants, produces both free 3'-OH and 5'-P termini randomly distributed along the DNA molecule, as suggested by labelling experiments with [alpha- 32P]dCTP and [gamma- 32 P]dATP. Moreover, when the reaction is carried out under low-salt conditions, cleavage is observed mainly at a specific site, located downstream of the ampicillin resistance gene (close to position 3200), ending with the deletion of a fragment of approximately 70 nucleotides. This cleavage pattern is similar to that obtained under the same conditions with mung bean nuclease, a single-strand endonuclease. Furthermore, pBR322 DNA treated with PD-L1 shows reduced transforming activity with E . coli HB101 competent cells in comparison to untreated control plasmid DNA.     

Concentrative uptake of cyclic ADP-ribose generated by BST-1+ stroma stimulates proliferation of human hematopoietic progenitors

Cyclic ADP-ribose (cADPR) is an intracellular calcium mobilizer generated from NAD(+) by the ADP-ribosyl cyclases CD38 and BST-1. cADPR, both exogenously added and paracrinally produced by a CD38(+) feeder layer, has recently been demonstrated to stimulate the in vitro proliferation of human hemopoietic progenitors (HP) and also the in vivo expansion of hemopoietic stem cells. The low density of BST-1 expression on bone marrow (BM) stromal cells and the low specific activity of the enzyme made it unclear whether cADPR generation by a BST-1(+) stroma could stimulate HP proliferation in the BM microenvironment. We developed and characterized two BST-1(+) stromal cell lines, expressing an ectocellular cyclase activity similar to that of BST-1(+) human mesenchymal stem cells, the precursors of BM stromal cells. Long term co-culture of cord blood-derived HP over these BST-1(+) feeders determined their expansion. Influx of paracrinally generated cADPR into clonogenic HP was mediated by a concentrative, nitrobenzylthioinosine- and dipyridamole-inhibitable nucleoside transporter, this providing a possible explanation to the effectiveness of the hormone-like concentrations of the cyclic nucleotide measured in the medium conditioned by BST-1(+) feeders. These results suggest that the BST-1-catalyzed generation of extracellular cADPR, followed by the concentrative uptake of the cyclic nucleotide by HP, may be physiologically relevant in normal hemopoiesis.     

Mechanism of hairpin-duplex conversion for the HIV-1 dimerization initiation site

We have used the dimerization initiation site of HIV-1 genomic RNA as a model to investigate hairpin-duplex interconversion with a combination of fluorescence, UV melting, gel electrophoresis, and x-ray crystallographic techniques. Fluorescence studies with molecular beacons and crystallization experiments with 23-nucleotide dimerization initiation site fragments showed that the ratio of hairpin to duplex formed after annealing in water essentially depends on RNA concentration and not on cooling kinetics. With natural sequences allowing to form the most stable duplex, and thus also the loop-loop complex (or "kissing complex"), concentrations as low as 3 mum in strands are necessary to obtain a majority of the hairpin form. With a mutated sequence preventing kissing complex formation, a majority of hairpins was even obtained at 80 mum in strands. However, this did not prevent an efficient conversion from hairpin to duplex in the presence of salts. Kinetic considerations are in favor of duplex formation from intermediates involving hairpins engaged in cruciform dimers rather than from free strands. The very first step of formation of such a cruciform intermediate could be trapped in a crystal structure. This mechanism might be significant for the dynamics of small RNAs beyond the strict field of HIV-1.     

Structure of minor oligosaccharides from the lipopolysaccharide fraction from Pseudomonas stutzeri OX1

A minor oligosaccharide fraction was isolated after complete de-acylation of the lipooligosaccharide extracted from Pseudomonas stutzeri OX1. The full structure of this oligosaccharide was obtained by chemical degradation, NMR spectroscopy and MALDI-TOF MS spectrometry. These experiments showed the presence of two novel oligosaccharides (OS1 and OS2): [structure: see text] where R=(S)-Pyr(-->4,6) in OS1 and alpha-Rha-(1-->3) in OS2. All sugars are D-pyranoses, except Rha, which is L-pyranose. Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, Pyr is pyruvic acid, P is phosphate.     

Purification and characterization of phycocyanin from the blue-green alga Aphanizomenon flos-aquae

Aphanizomenon flos-aquae (AFA) is a blue-green alga and represents a nutrient-dense food source. In this study the presence of phycocyanin (PC), a blue protein belonging to the photosynthetic apparatus, has been demonstrated in AFA. An efficient method for its separation has been set up: PC can be purified by a simple single step chromatographic run using a hydroxyapatite column (ratio A620/A280 of 4.78), allowing its usage for health-enhancing properties while eliminating other aspecific algal components. Proteomic investigation and HPLC analysis of purified AFA phycobilisomes revealed that, contrary to the well-characterized Synechocystis and Spirulina spp., only one type of biliprotein is present in phycobilisomes: phycocyanins with no allo-phycocyanins. Two subunit polypeptides of PC were also separated: the beta subunit containing two bilins as chromophore and the alpha subunit containing only one.     

Sarcoptic mange in wild carnivores and its co-occurrence with parasitic helminths in the Western Italian Alps

Between 2001 and 2004, 229 foxes, 36 stone martens and 48 badgers from the western Italian Alps were examined for sarcoptic mange and for gastrointestinal helminths to investigate their prevalence and geographical distribution and to point out the existence of potential interactions among them. Sarcoptic mange was observed in 25.3±2.8% SE of foxes and in 5.6±3.8% SE of stone martens, while no badger was found infected. Helminths belonged to Cestoidea Cyclophillidea (3.0±1.1% SE), Nematoda Trichurida (Capillaria aerophila and Trichuris vulpis: 6.5±1.6% SE; Trichinella britovi: 3.0±1.1% SE), Ascaridida (12.2±2.2% SE) and Strongylida (6.9±1.7% SE). Sarcoptic mange infection and the presence of helminths proved to be associated, with mangy foxes showing significantly higher prevalence of both cestode and nematode (particularly Ascaridida) worms. Moreover, considering three clusters of parasites (S. scabiei, nematodes and cestodes), more foxes than expected hosted simultaneously 2 and 3 taxa. These evidences suggest the existence of some kind of interaction, whose modalities and implications are discussed in this paper.     

Long-term trends of epilimnetic and hypolimnetic bacteria and organic carbon in a deep holo-oligomictic lake

We analysed the long-term dynamics (1980–2007) of hypolimnetic and epilimnetic bacterial abundances and organic carbon concentrations, both dissolved (DOC) and particulate (POC), in the deep holo-oligomictic Lake Maggiore, included in the Southern Alpine Lakes Long-Term Ecological Research (LTER) site. During the 28 years of investigation, bacterial abundance and POC concentrations did not decrease with declining phosphorus concentrations, while DOC concentrations showed a pronounced decrease in the epi- and hypolimnion. We used the annual mean total lake heat content and total annual precipitation as climate-related variables, and in-lake total phosphorus as a proxy for trophic state. The model (forward stepwise regression, FSR) showed that reduced anthropogenic pressure was more significant than climate change in driving the trend in DOC concentrations. Bacterial dynamics in the hypolimnion mirrored the fluctuations observed in the epilimnion, but average cell abundance was three times lower. The FSR model indicates that bacterial number variability was dependent on POC in the epilimnion and DOC in the hypolimnion. In the hypolimnion, cell biovolumes for rod and coccal morphotypes were significantly larger than in the epilimnion.