2,3-Dimethoxybenzo[i]phenanthridines: topoisomerase I-targeting anticancer agents

Appropriately substituted benzo[i]phenanthridines structurally related to nitidine, a benzo[c]phenanthridine alkaloid with antitumor activity, are active as topoisomerase I-targeting agents. Studies on benzo[i]phenanthridines have indicated analogues that possess a 2,3-methylenedioxy moiety and at least one and preferably two methoxyl groups at the 8- and 9-positions, such as 8,9-dimethoxy-2,3-methylenedioxybenzo[i]phenanthridine, 2, are active as topoisomerase I-targeting agents. Tetramethoxylated benzo[i]phenanthridines, wherein the 2,3-methylenedioxy moiety is replaced with methoxyl groups at the 2- and 3-position, are inactive as a topoisomerase I-targeting agent. These results initially suggested that the 2,3-methylenedioxy moiety was critical to the retention of potent activity. Further studies revealed that 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine, 7a, is more potent than 2 as a topoisomerase I-targeting agent. The observation that 2,3-dimethoxylated benzo[i]phenanthridines can actually exhibit enhanced activity prompted the present study in which several 8-substituted 2,3-dimethoxybenzo[i]phenanthridines were prepared and their pharmacological activities evaluated. The influence of NH(2), CN, CH(2)OH, OBn, OCH(3), OH, and NHCOCH(3 )substituents at the 8-position on the relative activity of these 2,3-dimethoxybenzo[i]phenanthridines was examined. Relative to these derivatives, 7a was the most potent topoisomerase I-targeting agent, possessing similar cytotoxicity to that of nitidine in the human lymphoblast tumor cell line, RPMI8402.     

Effects of bile acids on biliary epithelial cells: proliferation,cytotoxicity, and cytokine secretion

Hydrophobic bile acids, which are known to be cytotoxic for hepatocytes, are retained in high amount in the liver during cholestasis. Thus, we have investigated the effects of bile acids with various hydrophobicities on biliary epithelial cells. Biliary epithelial cells were cultured in the presence of tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), or taurolithocholate (TLC). Cell proliferation, viability, apoptosis and secretion of monocyte chemotactic protein-1 (MCP-1) and of interleukin-6 (IL-6) were studied. Cell proliferation was increased by TDC, and markedly decreased by TLC in a dose dependent manner (50-500 microM). Cell viability was significantly decreased by TLC and TCDC at 500 microM. TLC, TDC and TCDC induced apoptosis at high concentrations. The secretion of MCP-1 and IL-6 was markedly stimulated by TC. TUDC had no significant effect on any parameter. These findings demonstrate that hydrophobic bile acids were cytotoxic and induced apoptosis of biliary epithelial cells. Furthermore, TC, a major biliary acid in human bile, stimulated secretion of cytokines involved in the inflammatory and fibrotic processes occurring during cholestatic liver diseases.     

Effects of dietary soybean lecithin on plasma lipid transport and hepatic cholesterol metabolism in rats

Dietary lecithin can stimulate bile formation and biliary lipid secretion, particularly cholesterol output in bile. Studies also suggested that the lecithin-rich diet might modify hepatic cholesterol homeostasis and lipoprotein metabolism. Therefore, we examined hepatic activities of 3-hydroxy-3 methylglutaryl coenzyme A reductase "HMG -CoA reductase", cholesterol 7 alpha-hydroxylase and acyl-CoA: cholesterol acyltransferase "ACAT" as well as plasma lipids and lipoprotein composition in rats fed diets enriched with 20% of soybean lecithin during 14 days. We also evaluated the content of hepatic canalicular membrane proteins involved in lipid transport to the bile (all P-glycoproteins as detected by the C 219 antibody and the sister of P-glycoprotein "spgp" or bile acid export pump) by Western blotting. As predicted, lecithin diet modified hepatic cholesterol homeostasis. The activity of hepatic HMG-CoA reductase and cholesterol 7 alpha-hydroxylase was enhanced by 30 and 12% respectively, while microsomal ACAT activity showed a dramatic decrease of 75%. As previously reported from ACAT inhibition, the plasma level and size of very low-density lipoprotein (VLDL) were significantly decreased and bile acid pool size and biliary lipid output were significantly increased. The canalicular membrane content of lipid transporters was not significantly affected by dietary lecithin. The current data on inhibition of ACAT activity and related metabolic effects by lecithin mimic the previously reported effects following drug-induced inhibition of ACAT activity, suggesting potential beneficial effects of dietary lecithin supplementation in vascular disease.     

Hemisynthesis and preliminary evaluation of novel endocannabinoid analogues

Three new endocannabinoid analogues in which amide moiety was replaced either by oxomethylene group or ester moiety with simultaneous substitution of both alpha-hydrogens with methyl groups were synthesized and their abilities to interact with CB1-receptor and FAAH were investigated.     

Production, purification, and characterization of recombinant prohormone convertase 5 from baculovirus-infected insect cells

The discovery of the prohormone convertase (PC) family of enzymes has provided several good candidates (PC1, PC2, and PC5) for the enzymes responsible for the endoproteolytic cleavage of procholecystokinin (pro-CCK). Determination of the role of individual pro-hormone convertases in the processing of pro-CCK is complicated because several of these enzymes are found in endocrine tumor cells expressing CCK mRNA and in identified neurons in the brain. Production of active recombinant PC5 permits the determination of its ability to cleave substrates related to pro-CCK. Active PC5, secreted from baculovirus-infected Sf9 cells, was partially purified by ion-exchange chromatography. Western blot analysis confirmed the presence of the active form of the enzyme in infected cell media and its absence from uninfected cell media. The enzyme is most active at acidic pH 6.5 and is maximally activated by 5 mM calcium. PC5 was able to cleave both monobasic and dibasic substrates without a requirement for a basic residue at P-4 and it displayed a K(m) in the micromolar range. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and p-CMS, as well as by two specific PC inhibitors. This is the first reported preparation of active recombinant PC5. Like the other members of its family, it has the correct catalytic characteristics in vitro to play a role in the processing of neuropeptide precursor proteins into their final bioactive forms.     

Receptor kinase signalling in plants and animals: distinct molecular systems with mechanistic similarities

Plant genomes encode large numbers of receptor kinases that are structurally related to the tyrosine and serine/threonine families of receptor kinase found in animals. Here, we describe recent advances in the characterisation of several of these plant receptor kinases at the molecular level, including the identification of receptor complexes, small polypeptide ligands and cytosolic proteins involved in signal transduction and receptor downregulation. Phylogenetic analysis indicates that plant receptor kinases have evolved independently of the receptor kinase families found in animals. This hypothesis is supported by functional studies that have revealed differences between receptor kinase signalling in plants and animals, particularly concerning their interactions with cytosolic proteins. Despite these dissimilarities, however, plant and animal receptor kinases share many common features, such as their single membrane-pass structure, their inclusion in membrane-associated complexes, the involvement of dimerisation and trans autophosphorylation in receptor activation, and the existence of inhibitors and phosphatases that downregulate receptor activity. These points of convergence may represent features that are essential for a functional receptor-kinase signalling system.     

The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1^T for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis.     

Valbro : un nouveau site à vertébrés de l’Oligocène inférieur (MP22) de France (Quercy). I – Contexte géologique ; Mammalia : Rodentia,Hyaenodontida, Carnivora

The locality Valbro (Quercy, France) has yielded a rich fossil vertebrate fauna. Here are presented the geological context, a preliminary faunal list and the systematics of the Rodentia and carnivores, the most abundant taxa of the fauna. The Rodentia are known from 11 taxa: the Theridomyidae Blainvillimys gregarius Schlosser, 1884, Blainvillimys ?gemellus Vianey-Liaud, 1989, Issiodoromys medius (Vianey-Liaud, 1976) and S. cayluxi Schlosser, 1884, the Aplodontidae Plesispermophilus angustidens Filhol, 1882, the Gliridae Butseloglis tenuis (Bahlo, 1975), Butseloglis micio (Misonne, 1957) and Bransatoglis planus (Bahlo, 1975), the Sciuridae Palaeosciurus goti Vianey-Liaud, 1974 and cf. Oligopetes sp., and the Cricetidae Atavocricetodon sp. aff. A. nanus (Pelaez-Campomanes, 1995). Thirteen taxa of carnivores are present at Valbro; among the Hyaenodontida: Hyaenodon leptorhynchus Laizer et Parieu, 1838 and cf. Apterodon sp.; among the Carnivora: a Feliformia gen. et sp. indet., the Nimravidae Nimravus intermedius (Filhol, 1872), Dinailurictis bonali Helbing, 1922 and Nimravidae gen. et sp. indet. (Certainly belonging to one the two already identified species), the Ursida Pachycynodon crassirostris Schlosser, 1888, Pachycynodon sp., Amphicynodon sp. 1 cf. A. typicus (Schlosser, 1888), Amphicynodon ? sp. 2 and Ursida gen. et sp. indet. cf. Pachycynodon boriei (Filhol, 1876), the Mustelida M. olivieri Bonis, 1997, the Carnivora incertae sedis Palaeogale sectoria (Gervais, 1848–1852), the Arctoidea gen. et sp. indet. and Carnivora gen. et sp. indet. (probably representing taxa already identified at lower taxonomic levels). The evolutionary grade of B. gregarius of Valbro compares to that of the species from Mas de Got, La Plante 2 and Cavalé, which supports an MP22 age for Valbro. This datation is further supported by the association of Theridomyinae yielded by the locality (B. gregarius, B. gemellus, I. medius and S. cayluxi), and by the presence of a N. intermedius that well compares the material of the species from Villebramar, La Plante et Mas de Got, of D. bonali, P. sectoria, M. olivieri (species known only from Mas de Got), and by additional evidence from the remaining vertebrates from Valbro, especially the squamates. Despite a limited amount of specimens (341 specimens have been studied), the faunas of rodents and carnivores from Valbro are the most speciose and diverse known for the MP22 level and, for the Carnivora fauna in particular, for the Oligocene of the Old World.