Influence of substrate preference and complexity on co-existence of two non-native gammarideans (Crustacea: Amphipoda)

Substrate choice, swimming activity and risk to predation by burbot (Lota lota) of the well established Gammarus roeselii and the invader Dikerogammarus villosus were studied in mixed and single-species aquarium experiments. We used stones, gravel and aquatic weeds (Elodea, Chara) as substrates. We hypothesized that both species have different substrate preferences and that substrate affects the predation risk. We also assumed that presence of D. villosus influences substrate preference and predation risk of G. roeselii since the invader is known to affect the behavior of other gammarids. Adults of D. villosus in single species experiments and juveniles in mixed and single species experiments were evenly distributed over the different substrates but adults in mixed species experiments were more likely to prefer stone substrate. In contrast, adults and juveniles of G. roeselii clearly preferred aquatic weeds independent of the presence/absence of the invader. Both species preferred substrates with fissured surface over substrates with smooth surface. Gammarus roeselii was observed swimming more often than D. villosus in the open water but its swimming activity was lower when its preferred substrate was present compared with its swimming activity if non-preferred substrates were present. Predation rate of burbot on D. villosus was comparatively low and independent of the substrate. Burbot consumed many more G. roeselii than D. villosus, both in mixed and single species experiments. But when the preferred substrate of G. roeselii (weeds) was used in the experiments, predation rate of burbot on G. roeselii was somewhat lower than that when non-preferred substrates were present. The results of the experiments support our hypothesis that the gammarids studied here have different substrate preferences and that presence of the preferred substrate can affect predation risk. However, there is no evidence that presence of D. villosus affected substrate choice or predation risk in G. roeselii. We consider that differences in use of spatial niches permit co-existence of G. roeselii and D. villosus in the wild when substrates are diverse. The fact that G. roeselii than D. villosus is more often observed swimming in the open water may explain its higher risk of being captured by fish.     

Effect of (+)-usnic acid on mitochondrial functions as measured by mitochondria-specific oligonucleotide microarray in liver of B6C3F1 mice

Usnic acid is a lichen metabolite used as a weight-loss dietary supplement due to its uncoupling action on mitochondria. However, its use has been associated with severe liver disorders in some individuals. Animal studies conducted thus far evaluated the effects of usnic acid on mitochondria primarily by measuring the rate of oxygen consumption and/or ATP generation. To obtain further insight into usnic acid-mediated effects on mitochondria, we examined the expression levels of 542 genes associated with mitochondrial structure and functions in liver of B6C3F₁ female mice using a mitochondria-specific microarray. Beginning at 8 weeks of age, mice received usnic acid at 0, 60, 180, and 600 ppm in ground, irradiated 5LG6 diet for 14 days. Microarray analysis showed a significant effect of usnic acid on the expression of several genes only at the highest dose of 600 ppm. A prominent finding of the study was a significant induction of genes associated with complexes I through IV of the electron transport chain. Moreover, several genes involved in fatty acid oxidation, the Krebs cycle, apoptosis, and membrane transporters were over-expressed. Usnic acid is a lipophilic weak acid that can diffuse through mitochondrial membranes and cause a proton leak (uncoupling). The up-regulation of complexes I–IV may be a compensatory mechanism to maintain the proton gradient across the mitochondrial inner membrane. In addition, induction of fatty acid oxidation and the Krebs cycle may be an adaptive response to uncoupling of mitochondria.     

Human MutL-complexes monitor homologous recombination independently of mismatch repair

The role of mismatch repair proteins has been well studied in the context of DNA repair following DNA polymerase errors. Particularly in yeast, MSH2 and MSH6 have also been implicated in the regulation of genetic recombination, whereas MutL homologs appeared to be less important. So far, little is known about the role of the human MutL homolog hMLH1 in recombination, but recently described molecular interactions suggest an involvement. To identify activities of hMLH1 in this process, we applied an EGFP-based assay for the analysis of different mechanisms of DNA repair, initiated by a targeted double-stranded DNA break. We analysed 12 human cellular systems, differing in the hMLH1 and concomitantly in the hPMS1 and hPMS2 status via inducible protein expression, genetic reconstitution, or RNA interference. We demonstrate that hMLH1 and its complex partners hPMS1 and hPMS2 downregulate conservative homologous recombination (HR), particularly when involving DNA sequences with only short stretches of uninterrupted homology. Unexpectedly, hMSH2 is dispensable for this effect. Moreover, the damage-signaling kinase ATM and its substrates BLM and BACH1 are not strictly required, but the combined effect of ATM/ATR-signaling components may mediate the anti-recombinogenic effect. Our data indicate a protective role of hMutL-complexes in a process which may lead to detrimental genome rearrangements, in a manner which does not depend on mismatch repair.     

Mobilization and Acquisition of Sparingly Soluble P-Sources by Brassica Cultivars under P-Starved Environment Ⅱ. Rhizospheric pH changes, Redesigned Root Architecture and Pi-Uptake Kinetics

Non-mycorrhizal Brassica does not produce specialized root structures such as cluster or dauciform roots but is an effective user of P compared with other crops. In addition to P-uptake, utilization and remobilization activity, acquisition of orthophosphate (Pi) from extracellular sparingly P-sources or unavailable bound P-forms can be enhanced by biochemical rescue mechanisms such copious H~+-efflux and/or carboxylates exudation into rhizosphere by roots via plasmalemma H~+ ATPase and anion channels triggered by P-starvation. To visualize the dissolution of sparingly soluble Ca-phosphate (Ca-P), newly formed Ca-P was suspended in agar containing other essential nutrients. With NH_4~+ applied as the N source, the precipitate dissolved in the root vicinity can be ascribed to rhizosphere acidification, whereas no dissolution occurred with nitrate nutrition. To observe in situ rhizospheric pH changes, images were recorded after embedding the roots in agar containing bromocresol purple as a pH indicator. P-tolerant cultivar showed a greater decrease in pH than the sensitive cultivar in the culture media (the appearance of typical patterns of various colors of pH indicator in the root vicinity), and at stress P-level this acidification was more prominent. In experiment 2, low P-tolerant class-Ⅰ cultivars (Oscar and Con-Ⅱ) showed a greater decrease in solution media pH than low P-sensitive class-Ⅱ (Gold Rush and RL-18) cultivars, and P-contents of the cultivars was inversely related to decrease in culture media pH. To elucidate P-stressinduced remodeling and redesigning in a root architectural system, cultivars were grown in rhizoboxes in experiment 3.The elongation rates of primary roots increased as P-supply increased, but the elongation rates of the branched zones of primary roots decreased. The length of the lateral roots and topological index values increased when cultivars were exposed to a P-stress environment. To elucidate Pi-uptake kinetics, parameters related to P influx: maximal transport rate (V_(max)), the Michaelis-Menten constant (K_m), and the external concentration when net uptake is zero (C_(min)) were tested in experiment 4. Lower K_m and C_(min) values were better indicative of the P-uptake ability of the class-Ⅰ cultivars, evidencing their adaptability to P-starved environmental cues. In experiment 5, class-Ⅰ cultivars exuded two- to threefold more carboxylates than class-Ⅱ cultivars under the P-stress environment. The amount and types of carboxylates exuded from the roots of P-starved plants differed from those of plants grown under P-sufficient conditions. Nevertheless, the exudation rate of both class-Ⅰ and class-Ⅱ cultivars decreased with time, and the highest exudation rate was found after the first 4 h of carboxylates collection. Higher P uptake by class-Ⅰ cultivars was significantly related to the drop in root medium pH, which can be ascribed to H~+-efflux from the roots supplied with sparingly soluble rock-P and Ca_3(PO_4)_2. These classical rescue strategies provided the basis of P-solubilization and acquisition from sparingly soluble P-sources by Brassica cultivars to thrive in a typically stressful environment.     

Big dynorphin as a putative endogenous ligand for the kappa-opioid receptor

The diversity of peptide ligands for a particular receptor may provide a greater dynamic range of functional responses, while maintaining selectivity in receptor activation. Dynorphin A (Dyn A), and dynorphin B (Dyn B) are endogenous opioid peptides that activate the kappa-opioid receptor (KOR). Here, we characterized interactions of big dynorphin (Big Dyn), a 32-amino acid prodynorphin-derived peptide consisting of Dyn A and Dyn B, with human KOR, mu- (hMOR) and delta- (hDOR) opioid receptors and opioid receptor-like receptor 1 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dyn A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dyn B. Dyn A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn, while Dyn B demonstrated low selectivity. In contrast, Big Dyn activated G proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR-mediated activation of G proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of G proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of radioimmunoassay (RIA) and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid (CSF) with the ratio of Big Dyn and Dyn B being approximately 1:3. The presence in the brain implies that Big Dyn, along with other dynorphins, is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR-mediated activation of G proteins.     

Effect of substrate-dependent microbial ethylene production on plant growth

Various compounds have been identified as precursors/substrates for the synthesis of ethylene (C₂H₄) in soil. This study was designed to compare the efficiency of four substrates, namely L-methionine (L-MET), 2-keto-4-methylthiobutyric acid (KMBA), 1-aminocyclopropane-1-carboxylic acid (ACC), and calcium carbide (CaC₂), for ethylene biosynthesis in a sandy clay loam soil by gas chromatography. The classic “triple” response in etiolated pea seedling was employed as a bioassay to demonstrate the effect of substrate-dependent microbial production of ethylene on plant growth. Results revealed that an amendment with L-MET, KMBA, ACC (up to 0.10 g/kg soil) and CaC₂ (0.20 g/kg soil) significantly stimulated ethylene biosynthesis in soil. Overall, ACC proved to be the most effective substrate for ethylene production (1434 nmol/kg soil), followed by KMBA, L-MET, and CaC₂ in descending order. Results further revealed that ethylene accumulation in soil from these substrates caused a classic “triple” response in etiolated pea seedlings with different degrees of efficacy. A more obvious classic “triple” response was observed at 0.15, 0.10, and 0.20 g/kg soil of L-MET, KMBA/ACC, and CaC₂, respectively. Similarly, direct exposure of etiolated pea seedlings to commercial ethylene gas also modified the growth pattern in the same way. A significant direct correlation (r = 0.86 to 0.97) between substrate-derived C₂H₄ and the classic triple response in etiolated pea seedlings was observed. This study demonstrated that the presence of substrate(s) in soil may lead to increased ethylene concentration in the air of the soil, which may affect plant growth in a desired direction. Published in Russian in Mikrobiologiya, 2006, Vol. 75, No. 2, pp. 277–283. The text was submitted by the authors in English.     

Factors determining patient choice of device for GH therapy

AIM: To assess the factors determining patient choice of GH device, and whether offering free patient choice improves compliance with GH therapy. METHODS: A prospective cross-sectional study performed on patients offered free choice of GH device in a regional growth clinic. In a subgroup having home delivery, GH compliance was assessed using ampoule counts. RESULTS: 125 patients (74 (59%) male), median (range) 9.30 (1.0-18.3) years were commenced on GH from January 2001 to March 2004, and offered free choice of device. 68 (54%) chose a needled device, and 57 (46%) needle-free. There was no statistical difference in age, sex or diagnostic category between the two groups. Light blue devices were more likely to be chosen by males (p=0.056). Questionnaires giving reasons for choosing a device were available in 40, and a further 50 gave reasons for both choosing a specific device and not choosing others. Other than choice of needled/needle-free device, the factor most likely to determine choice was 'ease of use'. Only 6 (4.8%) subsequently changed device, and compliance remained high but unchanged at approximately 90%. CONCLUSIONS: There are no specific features which determine what GH device a patient will choose. For those units offering free patient choice, a wide range of different devices should be made available.     

Distribution of Holliday junctions and repair forks during Escherichia coli DNA double-strand break repair

Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites.     

A simple colorimetric method for screening cyclopiazonic acid in agricultural commodities

A simple and sensitive colorimetric method using p-dimethylaminobenzaldehyde was developed to detect cyclopiazonic acid in agricultural commodities. The method is sensitive within a range of 1–80 g/ml and allows screening large number of samples in a short time.