Carbon-Degrading Enzyme Activities Stimulated by Increased Nutrient Availability in Arctic Tundra Soils

Climate-induced warming of the Arctic tundra is expected to increase nutrient availability to soil microbes, which in turn may accelerate soil organic matter (SOM) decomposition. We increased nutrient availability via fertilization to investigate the microbial response via soil enzyme activities. Specifically, we measured potential activities of seven enzymes at four temperatures in three soil profiles (organic, organic/mineral interface, and mineral) from untreated native soils and from soils which had been fertilized with nitrogen (N) and phosphorus (P) since 1989 (23 years) and 2006 (six years). Fertilized plots within the 1989 site received annual additions of 10 g N⋅m^-2⋅year^-1 and 5 g P⋅m^-2⋅year^-1. Within the 2006 site, two fertilizer regimes were established – one in which plots received 5 g N⋅m^-2⋅year^-1 and 2.5 g P⋅m^-2⋅year^-1 and one in which plots received 10 g N⋅m^-2⋅year^-1 and 5 g P⋅m^-2⋅year^-1. The fertilization treatments increased activities of enzymes hydrolyzing carbon (C)-rich compounds but decreased phosphatase activities, especially in the organic soils. Activities of two enzymes that degrade N-rich compounds were not affected by the fertilization treatments. The fertilization treatments increased ratios of enzyme activities degrading C-rich compounds to those for N-rich compounds or phosphate, which could lead to changes in SOM chemistry over the long term and to losses of soil C. Accelerated SOM decomposition caused by increased nutrient availability could significantly offset predicted increased C fixation via stimulated net primary productivity in Arctic tundra ecosystems.     

Identification of the Argentine ant Linepithema humile (Hymenoptera: Formicidae) using an artificially synthesized trail pheromone and its effects on native Japanese ants

Applied Entomology and Zoology - Invasion by the Argentine ant Linepithema humile (Mayr) (Hymenoptera: Formicidae) causes the collapse of global ecosystems. Attempts to exterminate this invasive...     

Cold shock proteins improve E. coli cell-free synthesis in terms of soluble yields of aggregation-prone proteins

Protein folding is usually slowed-down at low temperatures, and thus low-temperature expression is an effective strategy to improve the soluble yield of aggregation-prone proteins. In this study, we investigated the effects of a variety of cold shock proteins and domains (Csps) on an Escherichia coli cell extract-based cell-free protein synthesis system (CF). Most of the 12 Csps that were successfully prepared dramatically improved the protein yields, by factors of more than 5 at 16°C and 2 at 23°C, to levels comparable to those obtained at 30°C. Their stimulatory effects were complementary to each other, while CspD and CspH were inhibitory. The Csps’ effects correlated well with their Pfam CSD family scores (PF00313.22). All of the investigated Csps, except CspH, similarly possessed RNA binding and chaperon activities and increased the messenger RNA amount irrespective of their effect, suggesting that the proper balance between these activities was required for the enhancement. Unexpectedly, the 5′-untranslated region of cspA was less effective as the leader sequence. Our results demonstrated that the use of the Csps presented in this study will provide a simple and highly effective strategy for the CF, to improve the soluble yields of aggregation-prone proteins.     

Treatment with β2‐Adrenoceptor Agonist in Vivo Induces Human Clock Gene,Per1, mRNA Expression in Peripheral Blood

This study examined whether in vivo exposure to a β₂‐adrenoceptor agonist, tulobuterol, induces human Period1 (hPer1) mRNA expression in cells from peripheral whole blood. In one experiment, oral tulobuterol was administered to five healthy volunteers at 22:00 h, while in another, a transdermally tulobuterol patch was applied to the same five subjects at 20:00 h. In each experiment, serum tulobuterol concentrations were measured at four time points, and total RNA was isolated from peripheral blood cells for determinations of hPer1 mRNA expression by real‐time polymerase chain reaction. Both the tulobuterol tablet and the transdermal patch increased hPer1 mRNA expression, suggesting that analyses of human peripheral blood cells could reliably represent peripheral clock gene mRNA expression in vivo.     

Root orientation can affect detection accuracy of ground-penetrating radar

Aim

Ground-penetrating radar (GPR) has been applied to detect coarse tree roots. The horizontal angle of a root crossing a scanning line is a factor that affects both root detection and waveform parameter values. The purpose of this study was to quantitatively evaluate the influence of root orientation (x, degree) on two major waveform parameters, amplitude area (A, dB × ns) and time interval between zero crossings (T, ns).

Methods

We scanned four diameter classes of dowels in a sandy bed as simulated roots using a 900 MHz antenna from multiple angles to clarify the relationships between the parameters and x.

Results

Angle x strongly affected reflection images and A values. The variation in A(x) fitted a sinusoidal waveform, whereas T was independent of x. The value of A scanning at 90° was estimated by A values of arbitrary x in two orthogonal transects. The sum of T in all reflected waveforms showed a significant linear correlation with dowel diameter.

Conclusions

We clarified that root orientation dramatically affected root detection and A values. The sum of T of all reflected waveforms was a suitable parameter for estimating root diameter. Applying grid transects can overcome the effects of root orientation.     

Focus Issue on Calcium Signaling: Analyses of a Gravistimulation-Specific Ca2+ Signature in Arabidopsis using Parabolic Flights

Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense changes in the gravity vector (gravistimulation) and regulate their growth direction accordingly. In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation, achieved by rotating the specimens under the ambient 1g of the Earth, is known to induce a biphasic (transient and sustained) increase in cytoplasmic calcium concentration ([Ca2+]_c). However, the [Ca2+]_c increase genuinely caused by gravistimulation has not been identified because gravistimulation is generally accompanied by rotation of specimens on the ground (1g), adding an additional mechanical signal to the treatment. Here, we demonstrate a gravistimulation-specific Ca²⁺ response in Arabidopsis seedlings by separating rotation from gravistimulation by using the microgravity (less than 10⁻⁴g) conditions provided by parabolic flights. Gravistimulation without rotating the specimen caused a sustained [Ca2+]_c increase, which corresponds closely to the second sustained [Ca2+]_c increase observed in ground experiments. The [Ca2+]_c increases were analyzed under a variety of gravity intensities (e.g. 0.5g, 1.5g, or 2g) combined with rapid switching between hypergravity and microgravity, demonstrating that Arabidopsis seedlings possess a very rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes (0.5g–2g) into Ca²⁺ signals on a subsecond time scale.Calcium ion (Ca²⁺) functions as an intracellular second messenger in many signaling pathways in plants (White and Broadley, 2003; Hetherington and Brownlee, 2004; McAinsh and Pittman, 2009; Spalding and Harper, 2011). Endogenous and exogenous signals are spatiotemporally encoded by changing the free cytoplasmic concentration of Ca²⁺ ([Ca2+]_c), which in turn triggers [Ca2+]_c-dependent downstream signaling (Sanders et al., 2002; Dodd et al., 2010). A variety of [Ca2+]_c increases induced by diverse environmental and developmental stimuli are reported, such as phytohormones (Allen et al., 2000), temperature (Plieth et al., 1999; Dodd et al., 2006), and touch (Knight et al., 1991; Monshausen et al., 2009). The [Ca2+]_c increase couples each stimulus and appropriate physiological responses. In the Ca²⁺ signaling pathways, the stimulus-specific [Ca2+]_c pattern (e.g. amplitude and oscillation) provide the critical information for cellular signaling (Scrase-Field and Knight, 2003; Dodd et al., 2010). Therefore, identification of the stimulus-specific [Ca2+]_c signature is crucial for an understanding of the intracellular signaling pathways and physiological responses triggered by each stimulus, as shown in the case of cold acclimation (Knight et al., 1996; Knight and Knight, 2000).Plants often exhibit biphasic [Ca2+]_c increases in response to environmental stimuli. Thus, slow cooling causes a fast [Ca2+]_c transient followed by a second, extended [Ca2+]_c increase in Arabidopsis (Arabidopsis thaliana; Plieth et al., 1999; Knight and Knight, 2000). The Ca²⁺ channel blocker lanthanum (La³⁺) attenuated the fast transient but not the following increase (Knight and Knight, 2000), suggesting that these two [Ca2+]_c peaks have different origins. Similarly, hypoosmotic shock caused a biphasic [Ca2+]_c increase in tobacco (Nicotiana tabacum) suspension-culture cells (Takahashi et al., 1997; Cessna et al., 1998). The first [Ca2+]_c peak was inhibited by gadolinium (Gd³⁺), La³⁺, and the Ca²⁺ chelator EGTA (Takahashi et al., 1997; Cessna et al., 1998), whereas the second [Ca2+]_c increase was inhibited by the intracellular Ca²⁺ store-depleting agent caffeine but not by EGTA (Cessna et al., 1998). The amplitude of the first [Ca2+]_c peak affected the amplitude of the second increase and vice versa (Cessna et al., 1998). These results suggest that even though the two [Ca2+]_c peaks originate from different Ca²⁺ fluxes (e.g. Ca²⁺ influx through the plasma membrane and Ca²⁺ release from subcellular stores, respectively), they are closely interrelated, showing the importance of the kinetic and pharmacological analyses of these [Ca2+]_c increases.Changes in the gravity vector (gravistimulation) could work as crucial environmental stimuli in plants and are generally achieved by rotating the specimens (e.g. +180°) in ground experiments. Use of Arabidopsis seedlings expressing apoaequorin, a Ca²⁺-reporting photoprotein (Plieth and Trewavas, 2002; Toyota et al., 2008a), has revealed that gravistimulation induces a biphasic [Ca2+]_c increase that may be involved in the sensory pathway for gravity perception/response (Pickard, 2007; Toyota and Gilroy, 2013) and the intracellular distribution of auxin transporters (Benjamins et al., 2003; Zhang et al., 2011). These two Ca²⁺ changes have different characteristics. The first transient [Ca2+]_c increase depends on the rotational velocity but not angle, whereas the second sustained [Ca2+]_c increase depends on the rotational angle but not velocity. The first [Ca2+]_c transient was inhibited by Gd³⁺, La³⁺, and the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid but not by ruthenium red (RR), whereas the second sustained [Ca2+]_c increase was inhibited by all these chemicals. These results suggest that the first transient and second sustained [Ca2+]_c increases are related to the rotational stimulation and the gravistimulation, respectively, and are mediated by distinct molecular mechanisms (Toyota et al., 2008a). However, it has not been demonstrated directly that the second sustained [Ca2+]_c increase is induced solely by gravistimulation; it could be influenced by other factors, such as an interaction with the first transient [Ca2+]_c increase (Cessna et al., 1998), vibration, and/or deformation of plants during the rotation.To elucidate the genuine Ca²⁺ signature in response to gravistimulation in plants, we separated rotation and gravistimulation under microgravity (μg; less than 10⁻⁴g) conditions provided by parabolic flight (PF). Using this approach, we were able to apply rotation and gravistimulation to plants separately (Fig. 1). When Arabidopsis seedlings were rotated +180° under μg conditions, the [Ca2+]_c response to the rotation was transient and almost totally attenuated in a few seconds. Gravistimulation (transition from μg to 1.5g) was then applied to these prerotated specimens at the terminating phase of the PF. This gravistimulation without simultaneous rotation induced a sustained [Ca2+]_c increase. The kinetic properties of this sustained [Ca2+]_c increase were examined under different gravity intensities (0.5g–2g) and sequences of gravity intensity changes (Fig. 2A). This analysis revealed that gravistimulation-specific Ca²⁺ response has an almost linear dependency on gravitational acceleration (0.5g–2g) and an extremely rapid responsiveness of less than 1 s.Open in a separate windowFigure 1.Diagram of the experimental procedures for applying separately rotation and gravistimulation to Arabidopsis seedlings. Rotatory stimulation (green arrow) was applied by rotating the seedlings 180° under μg conditions, and 1.5g 180° rotation gravistimulation (blue arrow) was applied to the prerotated seedlings after μg.Open in a separate windowFigure 2.Acceleration, temperature, humidity, and pressure in an aircraft during flight experiments. A, Accelerations along x, y, and z axes in the aircraft during PF. The direction of flight (FWD) and coordinates (x, y, and z) are indicated in the bottom graph. The inset shows an enlargement of the acceleration along the z axis (gravitational acceleration) during μg conditions lasting for approximately 20 s. B, Temperature, humidity, and pressure in the aircraft during PF. Shaded areas in graphs denote the μg condition.     

Modulation of chondrocyte migration and aggregation by insulin-like growth factor-1 in cultured cartilage

The effect of insulin-like growth factor-1 (IGF-1) on the behavior of rabbit chondrocytes in cultured collagen (CL) gels initially seeded with 2 × 10⁵ cells/ml was examined. On day 5, the frequency of migrating cells cultured in presence of 100 ng IGF-1/ml was 0.04, which was 54 % of the frequency in IGF-1-free culture. The presence of IGF-1 caused an increase in the frequency of dividing cells from 0.09 to 0.13. These results suggest that IGF-1 suppressed the migration of chondrocytes in the CL gels while stimulating cell division in the initial culture phase. The proteolytic migration of cells was thought to be suppressed by the down-regulation of membrane type 1 matrix metalloproteinase by IGF-1. This contributed to the formation of aggregates with spherical-shaped cells that produced collagen type II.     

Reduced Serum Selenium Concentration in Miscarriage Incidence of Indonesian Subjects

Selenium is an essential nutrient for human health, and maternal selenium concentration has been reported to be associated with pregnancy outcome. To further investigate the possible role of selenium (Se) in miscarriage, we conducted a case–control study to evaluate the correlations among selenium status, glutathione peroxidase activity, and spontaneous abortion. A total of 46 subjects with normal pregnancies and 25 subjects with spontaneous abortion were recruited, and their serum selenium concentrations and serum glutathione peroxidase activities were analyzed. The total serum selenium concentrations in subjects with normal pregnancies were significantly higher than those of subjects with spontaneous abortion; however, the glutathione peroxidase activities were similar in both groups. We further separated the subjects into smoking and nonsmoking groups, and the logistic regression analysis suggested that total serum selenium concentration, but not serum glutathione peroxidase activity or smoking, was significantly correlated with the incidence of miscarriage. The present study thus reaffirms that low serum selenium levels are associated with miscarriage and that selenium plays an important role in pregnancy maintenance.     

Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl₂. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.