Phosphorylation-regulated nucleocytoplasmic trafficking of internalized fibroblast growth factor-1

Fibroblast growth factor-1 (FGF-1), which stimulates cell growth, differentiation, and migration, is capable of crossing cellular membranes to reach the cytosol and the nucleus in cells containing specific FGF receptors. The cell entry process can be monitored by phosphorylation of the translocated FGF-1. We present evidence that phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC)delta. The phosphorylated FGF-1 is subsequently exported to the cytosol. A mutant growth factor where serine at the phosphorylation site is exchanged with glutamic acid, to mimic phosphorylated FGF-1, is constitutively transported to the cytosol, whereas a mutant containing alanine at this site remains in the nucleus. The export can be blocked by leptomycin B, indicating active and receptor-mediated nuclear export of FGF-1. Thapsigargin, but not leptomycin B, prevents the appearance of active PKCdelta in the nucleus, and FGF-1 is in this case phosphorylated in the cytosol. Leptomycin B increases the amount of phosphorylated FGF-1 in the cells by preventing dephosphorylation of the growth factor, which seems to occur more rapidly in the cytoplasm than in the nucleus. The nucleocytoplasmic trafficking of the phosphorylated growth factor is likely to play a role in the activity of internalized FGF-1.     

ASF/SF2 and SC35 regulate the glutamate receptor subunit 2 alternative flip/flop splicing

The properties of the glutamate receptor subunits 1-4 (GluR1-4) are influenced by the alternative splicing of two homologous and mutually exclusive exons flip and flop. The flip form is most abundant during early development, while the flop form is dominant in adults. From transfections with a GluR2 mini-gene we show that flip is the preferred splice form in all tested cell lines, but coexpression of the SR-proteins ASF/SF2 and SC35 increases the flop to flip splice ratio. The increased flop incorporation depends on ASF/SF2- and SC35-dependent enhancer elements located in the flop exon, which stimulate the splicing between the flop exon and the preceding exon 13.     

Effects of process stability on anaerobic biodegradation of LAS in UASB reactors

Anaerobic biodegradation of linear alkylbenzene sulfonates (LAS) was studied in upflow anaerobic sludge blanket (UASB) reactors operated under mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions. LAS C12 concentration in the influents was 10 mg.L(-1), and the hydraulic retention time in the reactors was 2 days. Adsorption of LAS C12 was assessed in an autoclaved control reactor and ceased after 115 days. The reactors were operated for a minimum of 267 days; 40-80% removal of LAS C12 was observed. A temperature reduction from 55 degrees C to 32 degrees C for 30 h resulted in process imbalance as indicated by increase of volatile fatty acids (VFA). The imbalance was much more intense in the LAS amended reactor compared with an unamended reactor. At the same time, the process imbalance resulted in discontinued LAS removal. This finding indicates that process stability is a key factor in anaerobic biological removal of LAS. After a recovery period, the removal of LAS resumed, providing evidence of biological anaerobic LAS degradation. The removal remained constant until termination of experiments in the reactor. Biodegradation of LAS in the mesophilic reactor was at the same level as in the thermophilic reactor under stable conditions.     

Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo

RNA interference (RNAi) has become an invaluable tool for functional genomics. A critical use of this tool depends on an understanding of the factors that determine the specificity and activity of the active agent, small interfering RNA (siRNA). Several studies have concluded that tolerance of mutations can be considerable and hence lead to off-target effects. In this study, we have investigated in vivo the toleration of wobble (G:U) mutations in high activity siRNAs against Flap Endonuclease 1 (Fen1) and Aquaporin-4 (Aqp4). Mutations in the central part of the antisense strand caused a pronounced decrease in activity, while mutations in the 5′ and 3′ends were tolerated very well. Furthermore, based on analysis of nine different mutated siRNAs with widely differing intrinsic activities, we conclude that siRNA activity can be significantly enhanced by wobble mutations (relative to mRNA), in the 5′ terminal of the antisense strand. These findings should facilitate design of active siRNAs where the target mRNA offers limited choice of siRNA positions.     

The diet of feral raccoon dog (<Emphasis Type="Italic">Nyctereutes procyonoides</Emphasis>) and native badger (<Emphasis Type="Italic">Meles meles</Emphasis>) and red fox (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) in Denmark

The raccoon dog (Nyctereutes procyonoides) is an East Asian Canid that has been introduced in Europe. Introduction of alien species is an increasing conservation issue. We examined the diet of a recently established raccoon dog population in Denmark by analysing stomach content in 249 carcasses collected in 2008–2016. Raccoon dog diet was compared to the diet of native badger (Meles meles) and red fox (Vulpes vulpes) in Denmark. The most common food for raccoon dogs were invertebrates (frequency of occurrence, FO 69%), small mammals (FO 68%), birds (FO 41%), fruits (FO 38%), amphibians (FO 36%) and carrions (FO 34%). The occurrence of invertebrates was highest during spring and summer, while fruits, cereals and carrions were eaten most often during autumn and winter. As expected, raccoon dog shared the major food categories with badger and red fox, but generally, it had a wider dietary niche. Overall, dietary overlap between raccoon dog and badger was 0.74 (Pianka index, O_jk). The dietary overlap with red fox was relatively high in all seasons, peaking in summer (O_jk 0.87) and dropping in winter (O_jk 0.79). Despite the dietary overlap between the alien racoon dog and native red fox and badger, the species may coexist due to partitioning of feeding habitats and/or because the red fox is limited by other factors, e.g. diseases and anthropogenic activities. The introduced raccoon dog seems to fit a dietary niche between badger and red foxes in human-dominated landscapes in north-western Europe.     

An Exported Inducer Peptide Regulates Bacteriocin Production in Enterococcus faecium CTC492

Production of the bacteriocins enterocin A and enterocin B in Enterococcus faecium CTC492 was dependent on the presence of an extracellular peptide produced by the strain itself. This induction factor (EntF) was purified, and amino acid sequencing combined with DNA sequencing of the corresponding gene identified it as a peptide of 25 amino acids. The gene encodes a prepeptide of 41 amino acids, including a 16-amino-acid leader peptide of the double-glycine type. Environmental factors influenced the level of bacteriocin production in E. faecium CTC492. The optimal pH for bacteriocin production was 6.2. At pH 5.5, growth was slow, and very little bacteriocin was formed. The presence of NaCl or ethanol (EtOH) was also inhibitory to bacteriocin production, and at high concentrations of these solutes, no bacteriocin production was observed. The induction factor induced its own synthesis, and by dilution of the culture 10⁶ times or more, nonproducing cultures were obtained. Bacteriocin production was induced in these cultures by addition of EntF. The response was linear, and low bacteriocin production could be induced by about 10⁻¹⁷ M EntF. This response was attenuated by low pH or the presence of high concentrations of NaCl or EtOH, and 300 times more EntF was needed to induce detectable bacteriocin production in the presence of 6.5% NaCl. High levels of bacteriocin production in cultures grown at low pH or in the presence of high concentrations of NaCl or EtOH were obtained by addition of sufficient amounts of EntF.     

Hydroxylated Analogues of 5-Aminovaleric Acid as 4-Aminobutyric AcidB Receptor Antagonists: Stereostructure-Activity Relationships

The (R) and (S) forms of 5-amino-2-hydroxyvaleric acid (2-OH-DAVA) and 5-amino-4-hydroxyvaleric acid (4-OH-DAVA) were designed as structural hybrids of the 4-aminobutyric acidB (GABAB) agonist (R)-(-)-4-amino-3-hydroxybutyric acid [(R)-(-)-3-OH-GABA] and the GABAB antagonist 5-aminovaleric acid (DAVA). (S)-(-)-2-OH-DAVA and (R)-(-)-4-OH-DAVA showed a moderately potent affinity for GABAB receptor sites in rat brain and showed GABAB antagonist effects in a guinea pig ileum preparation. The respective enantiomers, (R)-(+)-2-OH-DAVA and (S)-(+)-4-OH-DAVA, were markedly weaker in both test systems. All four compounds were weak inhibitors of GABAA receptor binding in rat brain, and none of them significantly affected synaptosomal GABA uptake. Based on molecular modeling studies it has been demonstrated that low-energy conformations of (R)-(-)-3-OH-GABA, (S)-(-)-2-OH-DAVA, and (R)-(-)-4-OH-DAVA can be superimposed. These conformations may reflect the shapes adopted by these conformationally flexible compounds during their interaction with GABAB receptors. The present studies emphasize the similar, but distinct, constraints imposed on agonists and antagonists for GABAB receptors.     

Resource availability,mating opportunity and sexual selection intensity influence the expression of male alternative reproductive tactics

The expression of alternative reproductive tactics can be plastic and occur simultaneously depending on cues that vary spatially or temporally. For example, variation in resources and sexual selection intensity is expected to influence the pay‐off of each tactic and shape the decision of which tactic to employ. Males of the nuptial gift‐giving spider Pisaura mirabilis can adopt three tactics: offering a genuine prey gift, a ‘worthless’ non‐nutritious gift or no gift. We hypothesized that resources and/or male body condition, and mating opportunity and sexual selection intensity, vary over the course of the mating season to shape the co‐existence of alternative traits. We measured these variables in the field over two seasons, to investigate the predictions that as the mating season progresses, (i) males become more likely to employ a gift‐giving tactic, and (ii) the likelihood of switching from worthless to genuine gifts increases. Prey availability increased over the season and co‐varied with the propensity of males to employ the gift‐giving tactic, but we found no support for condition‐dependent gift giving. Males responded to an increase in female availability by increasing their mating effort (gift production). Furthermore, the frequency of genuine gift use increased with sexual selection intensity, consistent with the assumption that sperm competition intensity increases with time. Our results suggest that the frequency of alternative tactics is shaped by seasonal changes in ecological factors and sexual selection. This leads to relaxed selection for the gift‐giving tactic early in the season when females are less choosy and resources more scarce, and increased selection for genuine gifts later in the season driven by mating opportunity and risk of sperm competition.