A phylogeny reconstruction of the Dendrophylliidae (Cnidaria,Scleractinia) based on molecular and micromorphological criteria,and its ecological implications

Recent molecular phylogenetic studies have shown that most traditional families of zooxanthellate shallow‐water scleractinians are polyphyletic, whereas most families mainly composed of deep‐sea and azooxanthellate species are monophyletic. In this context, the family Dendrophylliidae (Cnidaria, Scleractinia) has unique features. It shows a remarkable variation of morphological and ecological traits by including species that are either colonial or solitary, zooxanthellate or azooxanthellate, and inhabiting shallow or deep water. Despite this morphological heterogeneity, recent molecular works have confirmed that this family is monophyletic. Nevertheless, what so far is known about the evolutionary relationships within this family, is predominantly based on skeleton macromorphology, while most of its species have remained unstudied from a molecular point of view. Therefore, we analysed 11 dendrophylliid genera, four of which were investigated for the first time, and 30 species at molecular, micromorphological and microstructural levels. We present a robust molecular phylogeny reconstruction based on two mitochondrial markers (COI and the intergenic spacer between COI and 16S) and one nuclear (rDNA), which is used as basis to compare micromorphogical and microstructural character states within the family. The monophyly of the Dendrophylliidae is well supported by molecular data and also by the presence of rapid accretion deposits, which are ca. 5 μm in diameter and arranged in irregular clusters, and fibres that thicken the skeleton organized in small patches of a few micrometres in diameter. However, all genera represented by at least two species are not monophyletic, Tubastraea excluded. They were defined by traditional macromorphological characters that appear affected by convergence, homoplasy and intraspecific variation. Micromorphogical and microstructural analyses do not support the distinction of clades, with the exception of the organization of thickening deposits for the Tubastraea clade.     

Various sensitivities of yeasts to lycorine

Lycorine, an Amaryllidaceae alkaloid, is a powerful inhibitor of growth in higher plants and algae. Thirty-one strains of yeasts, belonging to different genera and species, were screened to study the effect of lycorine on their growth. The strains were incubated at 25 degrees C in a 2% glucose medium with different concentrations of lycorine (10, 50 and 100 microM), and their growth after 72 hours was evaluated. Most of the strains showed no sensitivity to lycorine. However, in Schizosaccharomyces pombe (IMAT-V Pbx) and Aureobasidium pullulans (DBV A77) lycorine significantly inhibited growth (59-73%), while, on the contrary, in Saccharomycopsis fibuligera (DBV 3812) and Cryptococcus terreus (CBS 1895) it was clearly stimulated (76-140%). The fact that lycorine inhibits growth in some yeasts while it stimulates it in others means that neither of the two previously formulated interpretations on the molecular mechanism of action of alkaloid can explain all cases. In other words, it does not seem that lycorine just inhibits protein synthesis, as claimed by Kukhanova et al. (1983), nor, on the other hand, do the data presented here prove that lycorine specifically inhibits ascorbic acid biosynthesis (Arrigoni et al., 1975). We must now check the ability of yeasts to split lycorine and study whether yeasts do actually have an ascorbic acid system.     

Acetylcholine esterase sensitivity to chronic administration of diphenylhydantoin and effects on cerebral enzymatic activities related to energy metabolism

The effect of chronic treatment (8 months) with diphenylhydantoin (DPH) on rat brain was studied. The activity of some enzymes related to energy transduction (lactate dehydrogenase, citrate synthase, and malate dehydrogenase; NADH-cytochromec reductase and cytochrome oxidase) and neurotransmission (acetylcholine esterase) was evaluated both in the whole brain homogenate and/or in the crude mitochondrial fraction. A clear-cut decrease of acetylcholine esterase activity was observed, the decrease continuing even after treatment was discontinued. Effects on energy metabolism and on lactate dehydrogenase, malate dehydrogenase, and cytochrome oxidase are discussed.     

Relationship between ascorbic acid and cell division

Proliferating cells require large amounts of ascorbic acid to reach cell division. The decrease in ascorbic acid caused by adding lycorine, an inhibitor of ascorbic acid biosynthesis, induces profound inhibition of cell division: the cell cycle is arrested in G1 and G2 phase, more than 90% of the cells being accumulated in G1 after some time. The effect of lycorine on mitotic index (MI) has been reversed by increasing experimentally the concentration of ascorbic acid in tissues. Ascorbic acid control on cell division is found to be specific, since isoascorbic acid is wholly ineffective. It is suggested that the principal role of ascorbic acid in the cell cycle may be related to its action in controlling the synthesis of hydroxyproline-containing proteins, which can be essential requirements for development of G1 and G2.     

The ascorbate system in recalcitrant and orthodox seeds

Recalcitrant seeds of Ginkgo biloba L., Quercus cerris L., Aesculus hippocastanum L. and Cycas revoluta Thunb. are shed by the plant at a high moisture content, contain a large amount of ascorbic acid (ASA) and maintain high ascorbate (ASC) peroxidase (EC 1.11.1.11) activity. Three proteins showing ASC peroxidase activity are present in G. biloba seeds. Conversely, dry orthodox seeds ( Vicia faba L., Avena sativa L., Pinus pinea L.) are completely devoid of ASA and ASC peroxidase. Experimentally induced rapid variations of the water level in both recalcitrant and orthodox seeds do not affect the ASC peroxidase; slow dehydration affects the ASC peroxidase activity moderately in recalcitrant seeds, but provokes a complete loss of germinability. Another peculiar difference between orthodox and recalcitrant seeds concerns the ascorbate recycling enzymes, ascorbate free radical (AFR) reductase (EC 1.6.5.4) and dehydroascorbate (DHA) reductase (EC 1.8.5.1). The DHA reduction capability is low in recalcitrant seeds, but is high in the orthodox ones. In contrast, AFR reductase activity is high in recalcitrant seeds and low in the orthodox ones. Data reported here concerning the ASC system appear to contribute to better understanding the recalcitrance. The presence of three different proteins showing ASC peroxidase activity in the archaic seed-bearing plant G. biloba and its involvement in the spermatophyte evolution is discussed.     

High confidence and sensitivity four-dimensional fractionation for human plasma proteome analysis

Reducing the complexity of plasma proteome through complex multidimensional fractionation protocols is critical for the detection of low abundance proteins that have the potential to be the most specific disease biomarkers. Therefore, we examined a four dimension profiling method, which includes low abundance protein enrichment, tryptic digestion and peptide fractionation by IEF, SCX and RP-LC. The application of peptide pI filtering as an additional criterion for the validation of the identifications allows to minimize the false discovery rate and to optimize the best settings of the protein identification database search engine. This sequential approach allows for the identification of low abundance proteins, such as angiogenin (10^?9 g/L), pigment epithelium growth factor (10^?8 g/L), hepatocyte growth factor activator (10^?7 g/L) and thrombospondin-1 (10^?6 g/L), having concentrations similar to those of many other growth factors and cytokines involved in disease pathophysiology.