Arabidopsis SOI33／AtENT8 Gene Encodes a Putative Equilibrative Nucleoside Transporter That Is Involved in Cytokinin Transport In Planta

The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transporters in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtlPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga22/atipt8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that S0133 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryodc cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and transzeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hypersensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of^3Hlabeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation in AtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.     

Analytical method for lipoperoxidation relevant reactive aldehydes in human sera by high-performance liquid chromatography–fluorescence detection

A validated, simple and sensitive HPLC method was developed for the simultaneous determination of lipoperoxidation relevant reactive aldehydes: glyoxal (GO), acrolein (ACR), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in human serum. The studied aldehydes were reacted with 2,2′-furil to form fluorescent difurylimidazole derivatives that were separated on a C₁₈ column using gradient elution and fluorescence detection at excitation and emission wavelengths of 250 and 355 nm, respectively. The method showed good linearity over the concentration ranges of 0.100–5.00, 0.200–10.0, 0.200–40.0, and 0.400–10.0 nmol/mL for GO, ACR, HNE, and MDA, respectively, with detection limits ranging from 0.030 to 0.11 nmol/mL. The percentage RSD of intraday and interday precision did not exceed 5.0 and 6.2%, respectively, and the accuracy (%found) ranged from 95.5 to 103%. The proposed method was applied for monitoring the four aldehydes in sera of healthy, diabetic, and rheumatic human subjects with simple pretreatment steps and without interference from endogenous components. By virtue of its high sensitivity and accuracy, our method enabled detection of differences between analytes concentrations in sera of human subjects under different clinical conditions.     

Presence of Neutrophil Extracellular Traps and Citrullinated Histone H3 in the Bloodstream of Critically Ill Patients

Neutrophil extracellular traps (NETs), a newly identified immune mechanism, are induced by inflammatory stimuli. Modification by citrullination of histone H3 is thought to be involved in the in vitro formation of NETs. The purposes of this study were to evaluate whether NETs and citrullinated histone H3 (Cit-H3) are present in the bloodstream of critically ill patients and to identify correlations with clinical and biological parameters. Blood samples were collected from intubated patients at the time of ICU admission from April to June 2011. To identify NETs, DNA and histone H3 were visualized simultaneously by immunofluorescence in blood smears. Cit-H3 was detected using a specific antibody. We assessed relationships of the presence of NETs and Cit-H3 with the existence of bacteria in tracheal aspirate, SIRS, diagnosis, WBC count, and concentrations of IL-8, TNF-α, cf-DNA, lactate, and HMGB1. Forty-nine patients were included. The median of age was 66.0 (IQR: 52.5–76.0) years. The diagnoses included trauma (7, 14.3%), infection (14, 28.6%), resuscitation from cardiopulmonary arrest (8, 16.3%), acute poisoning (4, 8.1%), heart disease (4, 8.1%), brain stroke (8, 16.3%), heat stroke (2, 4.1%), and others (2, 4.1%). We identified NETs in 5 patients and Cit-H3 in 11 patients. NETs and/or Cit-H3 were observed more frequently in “the presence of bacteria in tracheal aspirate” group (11/22, 50.0%) than in “the absence of bacteria in tracheal aspirate” group (4/27, 14.8%) (p<.01). Multiple logistic regression analysis showed that only the presence of bacteria in tracheal aspirate was significantly associated with the presence of NETs and/or Cit-H3. The presence of bacteria in tracheal aspirate may be one important factor associated with NET formation. NETs may play a pivotal role in the biological defense against the dissemination of pathogens from the respiratory tract to the bloodstream in potentially infected patients.     

Factors promoting maternal trophic egg provisioning in non-eusocial animals

The adaptive function of trophic egg-laying is generally regarded as extended parental investment to the offspring. However, the evolutionary factors promoting trophic egg-laying are still unclear, because the amount of maternal investment per offspring should be ideally equal between smaller offspring with trophic eggs and larger offspring without any additional investment. Several authors have suggested that trophic egg-laying should evolve only when egg size is constrained, but this hypothesis has not been evaluated. We investigated the evolutionary mechanisms of trophic egg-laying by two different approaches. First, we evaluated morphological constraints on egg size in two sibling ladybird species, Harmonia axyridis, which is known to produce trophic eggs, and H. yedoensis. Second, we theoretically predicted the optimal proportion of trophic eggs to total eggs and egg size in relation to environmental heterogeneity, predictability of environmental quality, and investment efficiency of trophic egg consumption. The intra- and interspecific morphological comparisons suggest that morphological constraints on the evolutionary determination of egg size are weak at best in the two ladybird species. Moreover, we theoretically showed that small egg size and trophic egg-laying are favoured in heterogeneous environments when mothers cannot adjust egg size plastically. We also showed that even a small reduction in investment efficiency makes a trophic egg strategy unlikely, despite relatively high environmental predictability. We conclude that trophic egg provisioning may be a flexible maternal adaptation to a highly heterogeneous environment rather than a response to a morphological constraint on egg size.     

Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.     

Your worst enemy could be your best friend: predator contributions to invasion resistance and persistence of natives

Native predators are postulated to have an important role in biotic resistance of communities to invasion and community resilience. Effects of predators can be complex, and mechanisms by which predators affect invasion success and impact are understood for only a few well-studied communities. We tested experimentally whether a native predator limits an invasive species’ success and impact on a native competitor for a community of aquatic insect larvae in water-filled containers. The native mosquito Aedes triseriatus alone had no significant effect on abundance of the invasive mosquito Aedes albopictus. The native predatory midge Corethrella appendiculata, at low or high density, significantly reduced A. albopictus abundance. This effect was not caused by trait-mediated oviposition avoidance of containers with predators, but instead was a density-mediated effect caused by predator-induced mortality. The presence of this predator significantly reduced survivorship of the native species, but high predator density also significantly increased development rate of the native species when the invader was present, consistent with predator-mediated release from interspecific competition with the invader. Thus, a native predator can indirectly benefit its native prey when a superior competitor invades. This shows the importance of native predators as a component of biodiversity for both biotic resistance to invasion and resilience of a community perturbed by successful invasion.     

The E3 ubiquitin ligase activity of Trip12 is essential for mouse embryogenesis

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12(mt/mt)) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12(mt/mt) embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16. In contrast, Trip12(mt/mt) ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12(mt/mt) ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.     

Secretion of inflammatory factors from chondrocytes by layilin signaling

Layilin (LAYN) is thought to be involved in reorganization of cytoskeleton structures, interacting with merlin, radixin, and talin. Also, LAYN is known to be one of the receptors for hyaluronic acid (HA).