An NADPH-dependent <Emphasis Type="Italic">Lactobacillus composti</Emphasis> short-chain dehydrogenase/reductase: characterization and application to (<Emphasis Type="Italic">R</Emphasis>)-1-phenylethanol synthesis

A new anti-Prelog short-chain dehydrogenase/reductase (SDR) encoding gene lcsdr was cloned from Lactobacillus composti DSM 18527, and heterologously expressed in Escherichia coli. LcSDR is nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and has a molecular weight of approximately 30 kDa. The optimal pH and temperature were 6.5 and 30?°C, respectively. The maximal reaction rate V_max was 133.9 U mg^?1; the Michaelis–Menten constant K _m of LcSDR were 0.345 mM for acetophenone (1a), and 0.085 mM for NADPH. Through introducing an EsGDH-catalyzed NADPH regeneration system, a biocatalytic process for (R)-1-phenylethanol ((R)-1b) was developed with outstanding time–space yield. Under the optimized conditions, 50 g l^?1 1a was converted to (R)-1b in 2 h with a yield of 93.8%, enantiomeric excess of product (e.e._p) above 99% and space–time yield of 562.8 g l^?1 d^?1.     

Antimicrobial activity,cytotoxicity and chemical analysis of lemongrass essential oil (<Emphasis Type="Italic">Cymbopogon flexuosus</Emphasis>) and pure citral

The aim of this study was to determine the antimicrobial effects of lemongrass essential oil (C. flexuosus) and to determine cytotoxic effects of both test compounds on human dermal fibroblasts. Antimicrobial susceptibility screening was carried out using the disk diffusion method. Antimicrobial resistance was observed in four of five Acinetobacter baumannii strains with two strains confirmed as multi-drug-resistant (MDR). All the strains tested were susceptible to both lemongrass and citral with zones of inhibition varying between 17 to 80 mm. The mean minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of citral (mic—0.14 % and mbc—0.3 % v/v) was lower than that of Lemongrass (mic—0.65 % and mbc—1.1 % v/v) determined using the microtitre plate method. Cell viability using human dermal fibroblasts (HDF; 106-05a) was determined following exposure to both compounds and a control (Grapeseed oil) using the XTT assay and the IC₅₀ determined at 0.095 % (v/v) for citral and 0.126 % (v/v) for lemongrass. Grapeseed oil had no effect on cell viability. Live cell imaging was performed using the LumaScope 500 imaging equipment and changes in HDF cell morphology such as necrotic features and shrinkage were observed. The ability of lemongrass essential oil (EO) and citral to inhibit and kill MDR A. baumannii highlights its potential for use in the management of drug-resistant infections; however, in vitro cytotoxicity does suggest further tests are needed before in vivo or ex vivo human exposure.     

Enantioconvergent hydrolysis of racemic styrene oxide at high concentration by a pair of novel epoxide hydrolases into (<Emphasis Type="Italic">R</Emphasis>)-phenyl-1,2-ethanediol

Objectives

To prepare (R)-phenyl-1,2-ethanediol ((R)-PED) with high enantiomeric excess (ee _p) and yield from racemic styrene oxide (rac-SO) at high concentration by bi-enzymatic catalysis.

Results

The bi-enzymatic catalysis was designed for enantioconvergent hydrolysis of rac-SO by a pair of novel epoxide hydrolases (EHs), a Vigna radiata EH3 (VrEH3) and a variant (AuEH2^A250I) of Aspergillus usamii EH2. The simultaneous addition mode of VrEH3 and AuEH2^A250I, exhibiting the highest average turnover frequency (aTOF) of 0.12 g h^?1 g^?1, was selected, by which rac-SO (10 mM) was converted into (R)-PED with 92.6% ee _p and 96.3% yield. Under the optimized reaction conditions: dry weight ratio 14:1 of VrEH3-expressing E. coli/vreh3 to AuEH2^A250I-expressing E. coli/Aueh2 ^A250I and reaction at 20 °C, rac-SO (10 mM) was completely hydrolyzed in 2.3 h, affording (R)-PED with 98% ee _p. At the weight ratio 0.8:1 of rac-SO to two mixed dry cells, (R)-PED with 97.4% ee _p and 98.7% yield was produced from 200 mM (24 mg/ml) rac-SO in 10.5 h.

Conclusions

Enantioconvergent hydrolysis of rac-SO at high concentration catalyzed by both VrEH3 and AuEH2^A250I is an effective method for preparing (R)-PED with high ee _p and yield.

     

The nitrite reductase activity of horse heart carboxymethylated-cytochrome <Emphasis Type="Italic">c</Emphasis> is modulated by cardiolipin

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4], whereas the value of k _on for NO₂ ^? reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M^?1 s^?1 (at pH 7.4). CL facilitates the NO₂ ^?-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k _on for the NO₂ ^?-mediated conversion of CL–CM-cytc-Fe(II) to CL–CM-cytc-Fe(II)-NO (5.6 ± 0.6 M^?1 s^?1; at pH 7.4) being slightly higher than that for the NO₂ ^?-mediated conversion of CL–cytc-Fe(II) to CL–cytc-Fe(II)-NO (2.6 ± 0.3 M^?1 s^?1; at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10^?6 M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k _on versus pH are ?1.05 ± 0.07 and ?1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH^?. These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL–CM-cytc.     

High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.     

Effects of adjuvant and conidial concentration on the efficacy of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> for the control of the two spotted spider mite, <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Greenhouse experiments were conducted on various crops (cucumber, tomato, eggplant, green bean) to ascertain the effects of Break-thru^® (polyether-polymethylsiloxane-copolymer, a silicone surfactant) and an oil emulsion, on Beauveria bassiana (Balsamo) Vuillemin (Bb) applications for the control of the two spotted spider mite, Tetranychus urticae Koch. The objectives were to compare a) the efficacy of Bb control when applied in aqueous Break-thru^® or an oil emulsion; b) the effects of various concentrations of Bb conidia, as affected by each surfactant; and c) the effects of Break-thru^® on the activity of the fungus. Conidia were suspended either in an aqueous Break-thru^® or an emulsifiable formulation at different conidial concentrations (1.05 × 10⁶, 2.1 × 10⁶ and 4.2 × 10⁶ conidia ml^?1) and sprayed onto leaves 2 weeks after artificial pest inoculation. Two sprays were performed, with an interval of one week from one spray to another, and T. urticae population counts (both motile and egg stages) were made on plant leaves 7 days after each spray. Bb conidia in Break-thru^® were more efficacious than conidia in emulsifiable formulation. With the highest rate of conidia (4.2 × 10⁶ conidia ml^?1), mortality of adult mites ranged from 60 ± 4.2 (mean ± SE) to 85.7 ± 4.3% in the Break-thru^® suspension and 39.4 ± 7 to 61.3 ± 6% in the oil emulsion. Leaf damage index was also substantially reduced from 70% in the unsprayed control to 40% by the application of Bb conidia at the highest rate with Break-thru^®. Break-thru^® can be combined with Bb in the integrated management of T. urticae and Isolate R444 is a promising candidate for the control of the pest.     

Inheritance and gene mapping of the white flower trait in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Despite being a unique marker trait, white flower inheritance in Brassica juncea remains poorly understood at the gene level. In this study, we investigated a B. juncea landrace with white petal in China. The white petal phenotype possessed defective chromoplasts with less plastoglobuli than the yellow petal phenotype. Genetic analysis confirmed that two independent recessive genes (Bjpc1 and Bjpc2) controlled the white flower trait. We then mapped the BjPC1 gene in a BC₄ population comprising 2295 individuals. We identified seven AFLP (amplified fragment length polymorphism) markers closely linked to the white flower gene. BLAST search revealed the sequence of AFLP fragments were highly homologous with the Scaffold000085 and Scaffold000031 sequences on the A02 chromosome in the Brassica rapa genome. Based on this sequence homology, we developed simple sequence repeat (SSR) primer pairs and identified 13 SSRs linked to the BjPC1 gene, including two that were co-segregated (SSR9 and SSR10). The two closest markers (SSR4 and SSR11) were respectively 0.9 and 0.4 cM on either side of BjPC1. BLAST analysis revealed that these marker sequences corresponded highly to A02 in B. juncea. They were mapped within a 33 kb genomic region on B. rapa A02 (corresponds to a 40 kb genomic region on B. juncea A02) that included three genes. Sequence BjuA008406, homologous to AtPES2 in Arabidopsis thaliana and Bra032956 in B. rapa, was the most likely candidate for BjPC1. These results should accelerate BjPC1 cloning and facilitate our understanding of the molecular mechanisms controlling B. juncea petal color.     

Using hydrothermal time concept to describe sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) seed germination response to temperature and water potential

To quantify both temperature (T) and water potential (ψ) effects on sesame (Sesamum indicum L.) seed germination (SG) and also to determine the cardinal T _s for this plant, a laboratory experiment was carried out using hydrothermal time model (HTT). For this purpose, four sesame cultivars (‘Asbomahalleh’, ‘Darab’, ‘Dashtestan’ and ‘Yellowhite’) were germinated at seven constant T _s (20, 25, 30, 35, 37, 39 and 43 °C) at each of the following ψ _s (0, ? 0.12, ? 0.24 and ? 0.36 MPa; provided by PEG 8000). Germination rate (GR) and germination percentage (GP) significantly influenced by ψ, T and their interactions in all cultivars (P ≤ 0.01). There was no significant difference, based on the confidence intervals of the model coefficients, between cultivars, so an average of cardinal T _s was 14.7, 35.4 and 47.2 °C for the minimum (T _b), optimum (T _o) and maximum (T _c) T _s, respectively, in the control condition (0 MPa). Hydrotime values in all cultivars decreased when T was increased to T _o and then remained constant at T _s > T _o (15 MPa h^?1). An average value of ψ _b(50) was estimated to be ? 1.23 MPa at T _s ≤ T _o and then increased linearly (0.1041 MPa°Ch^?1, the slope of the relationship between ψ _b(50) and supra-optimal T _s) with T when T _s increased above T _o and finally reached to zero at T _c. The T _b and T _o values were not influenced by ψ, but T _c value decreased (from 47.2 for zero to 43.5 °C for ? 0.36 MPa) at supra-optimal T _s as a result of the effect of ψ on GR. Based on our findings, this model (as a predictive tool) and or the estimated parameter values in this study can easily be used in sesame SG simulation models to quantitatively characterize the physiological status of sesame seed populations at different T _s and ψ _s.     

Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> GS1

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450_cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6β-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads’ superior resilience compared to E. coli. Whole-cell P. putida harboring P450_cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450_cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6β-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield Y_P/S of 79 %. To the authors’ knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.     

Impact of elevated CO<Subscript>2</Subscript> in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> rooted stem cuttings inoculated with <Emphasis Type="Italic">Frankia</Emphasis>

Impact of different levels of elevated CO ₂ on the activity of Frankia (Nitrogen-fixing actinomycete) in Casuarina equisetifolia rooted stem cuttings has been studied to understand the relationship between C. equisetifolia, Frankia and CO₂. The stem cuttings of C. equietifolia were collected and treated with 2000 ppm of Indole Butyric Acid (IBA) for rooting. Thus vegetative propagated rooted stem cuttings of C. equisetifolia were inoculated with Frankia and placed in the Open top chambers (OTC) with elevated CO₂ facilities. These planting stocks were maintained in the OTC for 12 months under different levels of elevated CO₂ (ambient control, 600 ppm, 900 ppm). After 12 months, the nodule numbers, bio mass, growth, and photosynthesis of C. equisetifolia rooted stem cuttings inoculated with Frankia were improved under 600 ppm of CO₂. The rooted stem cuttings of C. equisetifolia inoculated with Frankia showed a higher number of nodules under 900 ppm of CO₂ and cuttings without Frankia inoculation exhibited poor growth. Tissue Nitrogen (N) content was also higher under 900 ppm of CO₂ than ambient control and 600 ppm levels. The photosynthetic rate was higher (17.8 μ mol CO₂ m^?2 s^?1) in 900 ppm of CO₂ than in 600 ppm (13.2 μ mol CO₂ m^?2 s^?1) and ambient control (8.3 μ mol CO₂ m^?2 s^?1). This study showed that Frankia can improve growth, N fixation and photosynthesis of C. equietifolia rooted stem cuttings under extreme elevated CO₂ level conditions (900 ppm).     

Growth and uptake of caesium,rubidium, and potassium by ectomycorrhizal and saprotrophic fungi grown on either ammonium or nitrate as the N source

After the accident at the Fukushima Dai-ichi Nuclear Power Plant in 2011, high activities of radiocaesium have been reported in wild mushrooms in Japan. Fungi play an important role in the dynamics of radiocaesium in forest ecosystems. We examined the contents of caesium (Cs), rubidium (Rb), and potassium (K) in the mycelium of 15 isolates of ectomycorrhizal (EM) fungi and nine isolates of saprotrophic (SA) fungi in a synthetic medium with either ammonium chloride (NH₄Cl) or sodium nitrate (NaNO₃), supplemented with 1 ppm caesium chloride and rubidium chloride. The mycelia were harvested after 8 weeks of incubation, and the contents of Cs, Rb, and K were measured by inductively coupled plasma mass spectrometry. The dry weight of the mycelium in the medium with NH₄ was significantly higher than that with NO₃, although some EM species, Hebeloma, Astraeus, Scleroderma, and Pisolithus, grew well in the medium with NO₃. Among SA species, Crucibulum and Cyathus grew in the medium with NO₃. The uptakes of Cs, Rb, and K by Suillus, Pisolithus, and Rhizopogon were higher than that in other EM and SA species when they grew on the medium with NH₄, while the uptakes of these elements by Astraeus and Scleroderma were higher than those by other species grown on the medium with NO₃. The content of Rb was positively correlated with Cs (r = 0.85, p < 0.001) and K (r = 0.51, p < 0.001). The accumulation of Cs, Rb, and K was differently affected by the N source and fungal species.     

Exploring almond genetic variability useful for peach improvement: mapping major genes and QTLs in two interspecific almond × peach populations

Genetic analysis of a diverse set of 42 traits for flower (5), phenology (9), fruit quality (19), leaf (8) and disease resistance (1) was carried out in two interspecific almond × peach populations, an F₂ (T × E) and a BC₁ (T1E), from the cross between ‘Texas’ almond and ‘Earlygold’ peach. Traits related to flower, phenology, fruit quality, leaf morphology and resistance to powdery mildew were phenotyped over 3 years in two locations and studied for co-segregation with a large set of SNP and SSR markers. Three maps were used, one for the T × E and two for the T1E (T1E and E) population. Nine major genes were identified and mapped: anther color (Ag/ag and Ag2/ag2), flower color (Fc2/fc2), maturity date (MD/md), almond fruit type (almond vs. peach; Alf/alf), juiciness (Jui/jui), blood flesh (DBF2/dbf2), powdery mildew resistance (Vr3) and flower type (showy/non-showy; Sh/sh). These genes were often located in genome positions different from those for major genes for similar traits mapped before. Two of them explain fundamental aspects that define the fruit of peach with respect to that of almond: Alf and Jui, for its thick and juicy mesocarp, respectively. The genetics of quantitative traits was studied, and 32 QTLs were detected, with consistent behavior over the years. New alleles identified from almond for important traits such as red skin color, blood flesh, fruit weight and powdery mildew resistance may prove useful for the introduction of new variability into the peach gene pool used in commercial breeding programs.     

Exogenous application of thiamin promotes growth and antioxidative defense system at initial phases of development in salt-stressed plants of two maize cultivars differing in salinity tolerance

The effects of thiamin (Thi) applied as seed soaking or foliar spray on some key physiological parameters were investigated in two differentially salt-responsive maize (Zea mays L.) cultivars, DK 5783 and Apex 836 F1, exposed to saline stress in two different experiments. An initial experiment (germination experiment) was designed to identify appropriate doses of Thi which could lessen the deleterious effects of salt on plants and screen all available maize cultivars for their differential tolerance to salt stress (100 mM NaCl). The seeds of nine maize cultivars were soaked for 24 h in solutions containing six levels of Thi (25, 50, 75, 100, 125 and 150 mg l^?1). Based on the results obtained from the germination experiment, maize cultivar DK 5783 was found to be the most salt tolerant and Apex 836 as the most sensitive cultivar. Also, of six Thi levels used, two levels (100 and 125 mg l^?1) were chosen for subsequent studies. In the second experiment (glasshouse experiment), two maize cultivars, DK 5783 (salt tolerant) and Apex 836 (salt sensitive) were subjected to saline regime (100 mM NaCl) and two levels of Thi (100 and 125 mg l^?1) applied as foliar spray. Salt stress markedly suppressed shoot and root dry mass, total chlorophylls (“a” + “b”), leaf water potential and maximum fluorescence yield (Fv/Fm) in the plants of both maize cultivars, but it increased proline accumulation, leaf osmotic pressure, malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) concentrations, electrolyte leakage (EL) as well as activities of some key antioxidant enzymes, superoxide dismutase (SOD; EC. 1.15.1.1), peroxidase (POD; EC. 1.11.1.7) and catalase (CAT; EC. 1.11.1.6). Salt-induced reduction in plant growth parameters was higher in the salt-sensitive cultivar, Apex 836, which was found to be associated with relatively increased EL, and MDA and H₂O₂ levels, and decreased activities of the key antioxidant enzymes. Application of Thi as seed soaking or foliar spray partly mitigated the deleterious effects of salinity on plants of both maize cultivars. The most promising effect of Thi on alleviation of adverse effects of salt stress on maize plants was found when it was applied as foliar spray at 100 mg l^?1. Thiamin application considerably reduced tissue Na⁺ concentration, but improved those of N, P, Ca²⁺ and K⁺ in the salt-stressed maize plants. Exogenously applied thiamin-induced growth improvement in maize plants was found to be associated with reduced membrane permeability, MDA and H₂O₂ levels, and altered activities of some key antioxidant enzymes such as CAT, SOD and POD as well as increased photosynthetic pigment concentration under saline regime.     

Growth and nodulation in <Emphasis Type="Italic">Alnus sieboldiana</Emphasis> in response to <Emphasis Type="Italic">Frankia</Emphasis> inoculation and nitrogen treatments

Key message

We investigated a Frankia – Alnus sieboldiana symbiosis, including the minimum inoculum dose for constant nodulation, the period of time to nodulation after inoculation, and the effects of N on nodulation.

Abstract

Frankia is a nitrogen-fixing actinomycete that forms root nodules in some dicotyledonous plants, which are called actinorhizal. We studied nodule formation in Alnus sieboldiana, an actinorhizal plant, after inoculation with a Frankia isolate to establish techniques for Frankia inoculation and the cultivation of inoculated plants. Root nodules formed on seedlings of A. sieboldiana by 2 weeks after inoculation, and N₂ fixation measured by acetylene reduction activity started 3 weeks after inoculation. Nodulation was observed after inoculation with a Frankia isolate at 0.001 μL packed cell volume (pcv). The number of nodules formed on the seedlings inoculated with Frankia at more than 0.05 μL pcv was not significantly different. Nodule development and N₂ fixation were reduced when inoculated seedlings were treated weekly with 15 mM NH₄NO₃-N. In contrast, treatment with 3.75 or 0.9375 mM NH₄NO₃-N did not inhibit nodule development or N₂ fixation of inoculated seedlings by 15 weeks of N treatment.

     

The modulation of NMDA receptors and <Emphasis Type="SmallCaps">l</Emphasis>-arginine/nitric oxide pathway is implicated in the anti-immobility effect of creatine in the tail suspension test

The modulation of N-methyl-D-aspartate receptor (NMDAR) and l-arginine/nitric oxide (NO) pathway is a therapeutic strategy for treating depression and neurologic disorders that involves excitotoxicity. Literature data have reported that creatine exhibits antidepressant and neuroprotective effects, but the implication of NMDAR and l-arginine/nitric oxide (NO) pathway in these effects is not established. This study evaluated the influence of pharmacological agents that modulate NMDAR/l-arginine-NO pathway in the anti-immobility effect of creatine in the tail suspension test (TST) in mice. The NOx levels and cellular viability in hippocampal and cerebrocortical slices of creatine-treated mice were also evaluated. The anti-immobility effect of creatine (10 mg/kg, po) in the TST was abolished by NMDA (0.1 pmol/mouse, icv), d-serine (30 µg/mouse, icv, glycine-site NMDAR agonist), arcaine (1 mg/kg, ip, polyamine site NMDAR antagonist), l-arginine (750 mg/kg, ip, NO precursor), SNAP (25 μg/mouse, icv, NO donor), L-NAME (175 mg/kg, ip, non-selective NOS inhibitor) or 7-nitroindazole (50 mg/kg, ip, neuronal NOS inhibitor), but not by DNQX (2.5 µg/mouse, icv, AMPA receptor antagonist). The combined administration of sub-effective doses of creatine (0.01 mg/kg, po) and NMDAR antagonists MK-801 (0.001 mg/kg, po) or ketamine (0.1 mg/kg, ip) reduced immobility time in the TST. Creatine (10 mg/kg, po) increased cellular viability in hippocampal and cerebrocortical slices and enhanced hippocampal and cerebrocortical NO _x levels, an effect potentiated by l-arginine or SNAP and abolished by 7-nitroindazole or L-NAME. In conclusion, the anti-immobility effect of creatine in the TST involves NMDAR inhibition and enhancement of NO levels accompanied by an increase in neural viability.     

Characterization and fine mapping of <Emphasis Type="Italic">osh15</Emphasis>(<Emphasis Type="Italic">t</Emphasis>), a novel dwarf mutant gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plant height is one of the most important agronomic traits of plant architecture, and also affects grain yield in rice. In this study, we obtained a novel dwarf rice mutant of japonica variety Shennong9816, designated Shennong9816d. Compared with wild-type, the Shennong9816d plant height was significantly reduced, and the tiller number significantly increased. Additionally, the mutant yield component, and the number of large and small vascular bundles were significantly decreased compared with wild-type. Genetic analysis indicated that the Shennong9816d dwarf phenotype was controlled by a recessive nuclear gene, while the plant was shown to be sensitive to gibberellic acid. Using a large F₂ population derived from a cross between Shennong9816d and the indica rice variety Habataki, the osh15(t) gene was fine mapped between RM20891 and RM20898, within a physical distance of 73.78 kb. Sequencing analysis showed that Shennong9816d carries a 1 bp mutation and a 30 bp insertion in the OSH15 region. These results suggest that osh15(t) is a novel allelic mutant originally derived from japonica variety Shennong9816, which may be useful for introducing the semi-dwarf phenotype to improve plant architecture in rice breeding practice.     

A novel NADPH-dependent reductase of <Emphasis Type="Italic">Sulfobacillus acidophilus</Emphasis> TPY phenol hydroxylase: expression,characterization, and functional analysis

The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X ₄ -Cys-X ₂ -Cys-X _29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.