An Optimized Proteomics Approach Reveals Novel Alternative Proteins in Mouse Liver Development

Alternative ORFs (AltORFs) are unannotated sequences in genome that encode novel peptides or proteins named alternative proteins (AltProts). Although ribosome profiling and bioinformatics predict a large number of AltProts, mass spectrometry as the only direct way of identification is hampered by the short lengths and relative low abundance of AltProts. There is an urgent need for improvement of mass spectrometry methodologies for AltProt identification. Here, we report an approach based on size-exclusion chromatography for simultaneous enrichment and fractionation of AltProts from complex proteome. This method greatly simplifies the variance of AltProts discovery by enriching small proteins smaller than 40 kDa. In a systematic comparison between 10 methods, the approach we reported enabled the discovery of more AltProts with overall higher intensities, with less cost of time and effort compared to other workflows. We applied this approach to identify 89 novel AltProts from mouse liver, 39 of which were differentially expressed between embryonic and adult mice. During embryonic development, the upregulated AltProts were mainly involved in biological pathways on RNA splicing and processing, whereas the AltProts involved in metabolisms were more active in adult livers. Our study not only provides an effective approach for identifying AltProts but also novel AltProts that are potentially important in developmental biology.     

Effect of Different Carbon and Nitrogen Sources on Clostridium argentinense Toxigenicity in Coculture withPseudomonas mendocina

The effect of different carbon and nitrogen sources on the production of toxin by Clostridium argentinense was examined. The toxin production by C. argentinense in coculture with Pseudomonas mendocina increased in all the cases in relation to that produced by monocultures independent of the nature of the source. Using dextrin as carbon source C. argentinense produced the highest levels of toxin both in monocultures (300 LD₅₀/mL) and in cocultures with P. mendocina (5000 LD₅₀/mL). Experiments run in a microfermenter showed that the slow growth of cocultures associated with the assimilation of dextrin and the pH and Eh profiles favoured the production of toxin. Of the nitrogen sources assayed, corn steep liquor sustained the highest levels of toxin in both monocultures and cocultures with 3 and 2.8 fold increases with respect to that obtained using proteose peptone. The toxin production by C. argentinense cultures and C. argentinense–P. mendocina cocultures was highly dependent on the nature of the carbon and nitrogen sources used in the culture media. Growth of C. argentinense on substrates slowly assimilated stimulated the production of toxin.     

Cytokinin-binding proteins

This article is focused on the modalities of reception of cytokinins which remain largely unknown. It summarizes the main steps of the different protocols used to study cytokinin-binding proteins (CBPs). We place emphasis on the significance and specificity of the detection according to the properties of the probes used: radioactive or photoreactive cytokinins, fluorescent anticytokinins, anti-idiotype antibodies. The purification procedures are also examined. The cellular localisation and the putative physiological roles of the numerous and different CBPs found are considered. The interest of genetic and molecular studies is discussed.     

Corals shed bacteria as a potential mechanism of resilience to organic matter enrichment

Understanding the mechanisms of resilience of coral reefs to anthropogenic stressors is a critical step toward mitigating their current global decline. Coral–bacteria associations are fundamental to reef health and disease, but direct observations of these interactions remain largely unexplored. Here, we use novel technology, high-speed laser scanning confocal microscopy on live coral (Pocillopora damicornis), to test the hypothesis that corals exert control over the abundance of their associated bacterial communities by releasing (‘shedding'') bacteria from their surface, and that this mechanism can counteract bacterial growth stimulated by organic inputs. We also test the hypothesis that the coral pathogen Vibrio coralliilyticus can evade such a defense mechanism. This first report of direct observation with high-speed confocal microscopy of living coral and its associated bacterial community revealed a layer (3.3–146.8 μm thick) on the coral surface where bacteria were concentrated. The results of two independent experiments showed that the bacterial abundance in this layer was not sensitive to enrichment (5 mg l⁻¹ peptone), and that coral fragments exposed to enrichment released significantly more bacteria from their surfaces than control corals (P<0.01; 35.9±1.4 × 10⁵ cells cm⁻² coral versus 1.3±0.5 × 10⁵ cells cm⁻² coral). Our results provide direct support to the hypothesis that shedding bacteria may be an important mechanism by which coral-associated bacterial abundances are regulated under organic matter stress. Additionally, the novel ability to watch this ecological behavior in real-time at the microscale opens an unexplored avenue for mechanistic studies of coral–microbe interactions.     

Isolation and characterization of a human hepatic epithelial-like cell line (AKN-1) from a normal liver

Summary The isolation and characterization of human liver cell lines are rather difficult due to limited material and poor growth in cell culture. In this report, we present the isolation, culture and characterization of a new epithelial-like liver cell line (AKN-1) with a heterogeneous cell population and many characteristics of the biliary epithelium. The AKN-1 cell line stained positively with antibodies to epithelial cytokeratin polypetides CK 8, 18, and 19. In addition, the cell line expressed the anti-human epithelial-related antigen (MOC-31), the human epithelial antigen (HEA), and the gamma-glutamyl transpeptidase, the hematopoietic growth factor, stem cell factor, and also its receptor, c-kit. The cell line failed to express albumin and factor 8 by immunohistochemistry. It did show, however, a twofold increase in 7-ethoxyresorufin-O-deethylase activity. Cytogenetic characterization revealed rare breakpoints in chromosome 2, which to our knowledge, have not yet been reported in liver cells.     

Floral organogenesis of Chloranthus sessilifolius, with special emphasis on the morphological nature of the androecium of Chloranthus (Chloranthaceae)

 Floral organogenesis of Chloranthus sessilifolius K. F. Wu is described. The inflorescence primordium is dome-like in the beginning and then elongates, and bract primordia initiate almost decussately. Each floral primordium, arising from the axil of a bract, soon becomes a scale-like structure, with three primordia of androecial lobes originating from its abaxial part, and the gynoecial primordium in adaxial position. As the androecial lobes become more distinct, four thecae are already in differentiation, and the gynoecial primordium appears as a shallow disc. The androecial lobes do not extend their length until the thecae approach maturity and the stigma is differentiated. The androecial lobes are united at all the stages of development, and the entire androecium falls off as a unit at the end of anthesis. Based on these results, combined with published evidence from neobotany, palaeobotany and phylogenetic studies, the morphological nature of the androecium of Chloranthus is further discussed. Our studies support the viewpoint that the androecial structure of Chloranthus may have arisen by splitting of a single stamen with 2 marginal thecae. Received May 2, 2001 Accepted December 18, 2001     

Phylogeny of the Altingiaceae based on cpDNA matK, PY-IGS and nrDNA ITS sequences

 Phylogenetic relationships of the three genera of the family Altingiaceae, i.e., Altingia, Liquidambar and Semiliquidambar, based on matK sequences and the intergenic spacer between the psaA and ycf3 genes (PY-IGS) of cpDNA, and on the internal transcribed spacer (ITS) of nrDNA were studied. Phylogenetic trees based on the three data sets (matK, PY-IGS and ITS) were generated using Hamamelis japonica and Mytilaria laosensis (Hamamelidaceae), Cercidiphyllum japonicum (Cercidiphyllaceae), and Daphniphyllum calycinum (Daphniphyllaceae) as outgroups. The partition-homogeneity tests indicated that the three data sets and the combined data are homogeneous. A combined analysis also generated a strongly supported phylogeny. The phylogenetic trees show that the North American and western Asian species, L. styraciflua and L. orientalis, respectively, form a monophyletic group which is sister to the clade including all Asian species in the family. The genus Liquidambar is paraphyletic with Altingia and Semiliquidambar nested within. Phylogenetic analyses of the molecular data indicate that taxonomic reexamination of the generic delimitation in the Altingiaceae is needed. Received December 20, 2000 Accepted June 25, 2001     

Survival costs of reproduction vary with age in North American red squirrels

The costs of reproduction are expected to be higher under unfavourable conditions, so that breeding in years of low food supply should have important costs. In addition, the costs of reproduction may be contingent on the age of individuals, and young growing and old senescent individuals should suffer higher costs than the prime-age ones. We tested these predictions by investigating the costs of reproduction as a function of food availability and age in female North American red squirrels using the long-term data on survival and reproduction. We found that the costs of reproduction were independent of food supply, and we did not detect any trade-off between the current and future reproduction. We also did not detect any survival cost of reproduction for the prime-age females, but found evidence for survival costs in yearlings and old (6 years or above) females with successfully breeding individuals having a lower chance of survival compared with unsuccessful or non-breeding ones. These results supported our prediction that the costs of reproduction depended on the age of female red squirrels and were higher in young growing and old senescent individuals. Our study also indicated that, in contrast to large herbivores, heterogeneity in individual quality and viability selection in red squirrels do not affect the study of trade-offs and of the age variation in life-history traits.