Characterization of an acid inducible lipase Rv3203 from Mycobacterium tuberculosis H37Rv

The Rv3203 (LipV) of Mycobacterium tuberculosis (Mtb) H37Rv, is annotated as a member of Lip family based on the presence of characteristic consensus esterase motif ‘GXSXG’. In vitro culture studies of Mtb H37Ra indicated that expression of Rv3203 gene was up-regulated during acidic stress as compared to normal whereas no expression was observed under nutrient and oxidative stress conditions. Therefore, detailed characterization of Rv3203 was done by gene cloning and its further expression and purification as his-tagged protein in microbial expression system. The enzyme was purified to homogeneity by affinity chromatography. It demonstrated broad substrate specificity and preferentially hydrolyzed p-nitrophenyl myristate. The purified enzyme demonstrated an optimum activity at pH 8.0 and temperature 50 °C. The specific activity, K _m and V _max of enzyme was determined to be 21.29 U mg^?1 protein, 714.28 μM and 62.5 μmol ml^?1 min^?1, respectively. The pH stability assay and circular dichroism spectroscopic analysis revealed that Rv3203 protein is more stable in acidic condition. Tetrahydrolipstatin, a specific lipase inhibitor and RHC80267, a diacylglycerol lipase inhibitor abolished the activity of this enzyme. The catalytic triad residues were determined to be Ser⁵⁰, Asp¹⁸⁰ and His²⁰³ residues by site-directed mutagenesis.     

Promising properties of a formate dehydrogenase from a methanol-assimilating yeast Ogataea parapolymorpha DL-1 in His-tagged form

The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s^?1, and 2.83 mM^?1 s^?1, respectively. The K _m for NAD⁺ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.     

Peptide transport in yeast: uptake of radioactive trimethionine in Saccharomyces cerevisiae.

The synthesis of Met-Met-[¹⁴C]Met and [³H]Ac-Met-Met-Met are reported. Uptake of these compounds by Saccharomyces cerevisiae is examined in detail. The tripeptide Met-Met-Met is taken up by S. cerevisiae by a saturable system that is pH and temperature dependent and exhibits an apparent K_m of 7.7 × 10^?5m. Neither methionine nor dimethionine effectively compete with (Met)₃ uptake. The tripeptides Met-Ala-Met, Met-Met-Leu, (Ala)₃, (Leu)₃ and l-Met-l-Met-l-Met-OMe compete with (Met)₃ uptake whereas Ac(Met)₃, d-Met-d-Met-d-Met-OMe, (Lys)₃, and Gly-Met-Gly do not. Metabolic inhibitors (sodium azide, dinitrophenol, and potassium cyanide) completely prevent (Met)₃ uptake. Trimethionine is rapidly hydrolyzed by cellular peptidases after entry into the cell. Ac(Met)₃ is transported by a system that is pH and temperature dependent with an apparent K_m of 16 × 10^?5m.     

Enrichment and physiological characterization of an anaerobic ammonium-oxidizing bacterium ‘Candidatus Brocadia sapporoensis’

We successfully enriched a novel anaerobic ammonium-oxidizing (anammox) bacterium affiliated with the genus ‘Candidatus Brocadia’ with high purity (>90%) in a membrane bioreactor (MBR). The enriched bacterium was distantly related to the hitherto characterized ‘Ca. Brocadia fulgida’ and ‘Ca. Brocadia sinica’ with 96% and 93% of 16S ribosomal RNA gene sequence identity, respectively. The bacterium exhibited the common structural features of anammox bacteria and produced hydrazine in the presence of hydroxylamine under anoxic conditions. The temperature range of anammox activity was 20–45 °C with a maximum activity at 37 °C. The maximum specific growth rate (μ_max) was 0.0082 h^?1 at 37 °C, corresponding to a doubling time of 3.5 days. The half-saturation constant (K_S) for nitrite was 5 ± 2.5 μM. The anammox activity was inhibited by nitrite (IC₅₀ = 11.6 mM) but not by formate and acetate. The major respiratory quinone was identified to be menaquinone-7 (MK-7). The enriched anammox bacterium shared nearly half of genes with ‘Ca. Brocadia sinica’ and ‘Ca. Brocadia fulgida’. The enriched bacterium showed all known physiological characteristics of anammox bacteria and can be distinguished from the close relatives by its 16S rRNA gene sequence. Therefore, we proposed the name ‘Ca. Brocadia sapporoensis’ sp. nov.     

Characterization of EstB,a novel cold-active and organic solvent-tolerant esterase from marine microorganism Alcanivorax dieselolei B-5(T)

A novel esterase gene, estB, was cloned from the marine microorganism Alcanivorax dieselolei B-5(T) and overexpressed in E. coli DE3 (BL21). The expressed protein EstB with a predicted molecular weight of 45.1 kDa had a distinct catalytic triad (Ser²¹¹-Trp³⁵³-Gln³⁸⁵) and the classical consensus motif conserved in most lipases and esterases Gly²⁰⁹-X-Ser²¹¹-X-Gly²¹³. EstB showed very low similarity to any known proteins and displayed the highest similarity to the hypothetical protein (46 %) from Rhodococcus jostii RHA1. EstB showed the optimal activity around pH 8.5 and 20 °C and was identified to be extremely cold-adaptative retaining more than 95 % activity between 0 and 10 °C. The values of kinetic parameters on p-NP caproate (K _m, K _cat and K _cat/K _m) were 0.15 mM, 0.54 × 10³ s^?1 and 3.6 × 10³ s^?1 mM^?1, respectively. In addition, EstB showed remarkable stability in several studied organic solvents and detergents of high concentrations with the retention of more than 70 % activity after treatment for 30 min. The cold activity and its tolerance towards organic solvents made it a promising biocatalyst for industrial applications under extreme conditions.     

Subcellular Localization and Kinetic Characterization of a Gill (Na+, K+)-ATPase from the Giant Freshwater Prawn Macrobrachium rosenbergii

The stimulation by Mg²⁺, Na⁺, K⁺, NH₄ ⁺, and ATP of (Na⁺, K⁺)-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na⁺, K⁺)-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M _r. Under saturating Mg²⁺, Na⁺, and K⁺ concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V _M = 115.0 ± 2.3 U mg^?1, K _0.5 = 0.10 ± 0.01 mmol L^?1. Stimulation by Na⁺ (V _M = 110.0 ± 3.3 U mg^?1, K _0.5 = 1.30 ± 0.03 mmol L^?1), Mg²⁺ (V _M = 115.0 ± 4.6 U mg^?1, K _0.5 = 0.96 ± 0.03 mmol L^?1), NH₄ ⁺ (V _M = 141.0 ± 5.6 U mg^?1, K _0.5 = 1.90 ± 0.04 mmol L^?1), and K⁺ (V _M = 120.0 ± 2.4 U mg^?1, K _M = 2.74 ± 0.08 mmol L^?1) followed single saturation curves and, except for K⁺, exhibited site–site interaction kinetics. Ouabain inhibited ATPase activity by around 73 % with K _I = 12.4 ± 1.3 mol L^?1. Complementary inhibition studies suggest the presence of F₀F₁–, Na⁺-, or K⁺-ATPases, but not V(H⁺)- or Ca²⁺-ATPases, in the gill microsomal preparation. K⁺ and NH₄ ⁺ synergistically stimulated enzyme activity (≈25 %), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH₄ ⁺, and K⁺ of the gill enzyme.     

A chimeric α-amylase engineered from <Emphasis Type="Italic">Bacillus acidicola</Emphasis> and G<Emphasis Type="Italic">eobacillus thermoleovorans</Emphasis> with improved thermostability and catalytic efficiency

The α-amylase (Ba-amy) of Bacillus acidicola was fused with DNA fragments encoding partial N- and C-terminal region of thermostable α-amylase gene of Geobacillus thermoleovorans (Gt-amy). The chimeric enzyme (Ba-Gt-amy) expressed in Escherichia coli displays marked increase in catalytic efficiency [K _cat: 4 × 10⁴ s^?1 and K _cat/K _m: 5 × 10⁴ mL^?1 mg^?1 s^?1] and higher thermostability than Ba-amy. The melting temperature (T _m) of Ba-Gt-amy (73.8 °C) is also higher than Ba-amy (62 °C), and the CD spectrum analysis revealed the stability of the former, despite minor alteration in secondary structure. Langmuir–Hinshelwood kinetic analysis suggests that the adsorption of Ba-Gt-amy onto raw starch is more favourable than Ba-amy. Ba-Gt-amy is thus a suitable biocatalyst for raw starch saccharification at sub-gelatinization temperatures because of its acid stability, thermostability and Ca²⁺ independence, and better than the other known bacterial acidic α-amylases.     

Stereospecific oxidation of calmodulin by methionine sulfoxide reductase A

Methionine sulfoxide reductase A has long been known to reduce S-methionine sulfoxide, both as a free amino acid and within proteins. Recently the enzyme was shown to be bidirectional, capable of oxidizing free methionine and methionine in proteins to S-methionine sulfoxide. A feasible mechanism for controlling the directionality has been proposed, raising the possibility that reversible oxidation and reduction of methionine residues within proteins is a redox-based mechanism for cellular regulation. We undertook studies aimed at identifying proteins that are subject to site-specific, stereospecific oxidation and reduction of methionine residues. We found that calmodulin, which has nine methionine residues, is such a substrate for methionine sulfoxide reductase A. When calmodulin is in its calcium-bound form, Met77 is oxidized to S-methionine sulfoxide by methionine sulfoxide reductase A. When methionine sulfoxide reductase A operates in the reducing direction, the oxidized calmodulin is fully reduced back to its native form. We conclude that reversible covalent modification of Met77 may regulate the interaction of calmodulin with one or more of its many targets.     

The combined impact of metabolic gene polymorphisms on elite endurance athlete status and related phenotypes

Endurance performance is a complex phenotype subject to the influence of both environmental and genetic factors. Although the last decade has seen a variety of specific genetic factors proposed, many in metabolic pathways, each is likely to make a limited contribution to an ‘elite’ phenotype: it seems more likely that such status depends on the simultaneous presence of multiple such variants. The aim of the study was to investigate individually and in combination the association of common metabolic gene polymorphisms with endurance athlete status, the proportion of slow-twitch muscle fibers and maximal oxygen consumption. A total of 1,423 Russian athletes and 1,132 controls were genotyped for 15 gene polymorphisms, of which most were previously reported to be associated with athlete status or related intermediate phenotypes. Muscle fiber composition of m. vastus lateralis in 45 healthy men was determined by immunohistochemistry. Maximal oxygen consumption of 50 male rowers of national competitive standard was determined during an incremental test to exhaustion on a rowing ergometer. Ten ‘endurance alleles’ (NFATC4 Gly160, PPARA rs4253778 G, PPARD rs2016520 C, PPARGC1A Gly482, PPARGC1B 203Pro, PPP3R1 promoter 5I, TFAM 12Thr, UCP2 55Val, UCP3 rs1800849 T and VEGFA rs2010963 C) were first identified showing discrete associations with elite endurance athlete status. Next, to assess the combined impact of all 10 gene polymorphisms, all athletes were classified according to the number of ‘endurance’ alleles they possessed. The proportion of subjects with a high (≥9) number of ‘endurance’ alleles was greater in the best endurance athletes compared with controls (85.7 vs. 37.8%, P = 7.6 × 10^?6). The number of ‘endurance’ alleles was shown to be positively correlated (r = 0.50; P = 4.0 × 10^?4) with the proportion of fatigue-resistant slow-twitch fibers, and with maximal oxygen consumption (r = 0.46; P = 7.0 × 10^?4). These data suggest that the likelihood of becoming an elite endurance athlete depends on the carriage of a high number of endurance-related alleles.     

Directed evolution of GH43 β-xylosidase XylBH43 thermal stability and L186 saturation mutagenesis

Directed evolution of β-xylosidase XylBH43 using a single round of gene shuffling identified three mutations, R45K, M69P, and L186Y, that affect thermal stability parameter K _t ^0.5 by ?1.8 ± 0.1, 1.7 ± 0.3, and 3.2 ± 0.4 °C, respectively. In addition, a cluster of four mutations near hairpin loop-D83 improved K _t ^0.5 by ~3 °C; none of the individual amino acid changes measurably affect K _t ^0.5 . Saturation mutagenesis of L186 identified the variant L186K as having the most improved K _t ^0.5 value, by 8.1 ± 0.3 °C. The L186Y mutation was found to be additive, resulting in K _t ^0.5 increasing by up to 8.8 ± 0.3 °C when several beneficial mutations were combined. While k _cat of xylobiose and 4-nitrophenyl-β-d-xylopyranoside were found to be depressed from 8 to 83 % in the thermally improved mutants, K _m, K _ss (substrate inhibition), and K _i (product inhibition) values generally increased, resulting in lessened substrate and xylose inhibition.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

Determining the fluxes of Tl+ and K+ at the root surface of wheat and canola using Tl(I) and K ion-selective microelectrodes

The objectives of this study were to develop and evaluate a Tl⁺ ion-selective microelectrode (ISME) and to determine the basis for observed differences in Tl accumulation by durum wheat (Triticum turgidum L. var ‘Kyle’) and spring canola (Brassica napus L. cv ‘Hyola 401’). Seedlings were grown hydroponically and fluxes of K⁺ and Tl⁺ were measured at the root surface in solutions containing 5 μM Tl⁺ or 3 mM K⁺. After testing two different Tl(I) ionophores, a functional Tl⁺ ISME was developed from calix[4]arene tetra-n-propyl ether which had a detection limit of 2.5 µM and a slope of 56.6 mV/dec. Measurements of Tl⁺ flux indicate that Tl⁺ efflux occurred within 300–500 µm of the root tip, and influx farther from the root tip. Compared with canola, wheat had a slightly larger region of efflux and a smaller region of maximal influx, resulting in flux per root branch that was 2.3 to 4 times greater in canola than in wheat. The magnitude and pattern of K⁺ fluxes by the two species were more similar. Our results indicate that observed differences in Tl accumulation by wheat and canola are due both to differences in the magnitude of Tl flux per root branch of these species, and to differences in root morphology resulting in more root tips in canola than in wheat roots.     

1H, 13C and 15N resonance assignment of a zinc-binding methionine sulfoxide reductase type-B from the thermophilic archeabacterium Methanothermobacter thermoautotrophicus

The ¹H, ¹³C and ¹⁵N resonance assignment of a type-B zinc-binding methionine sulfoxide reductase lacking a ‘recycling’ cysteine from the thermophilic archeabacterium Methanothermobacter thermoautotrophicus is reported.     

Biodegradation and metabolic pathway of sulfamethoxazole by Pseudomonas psychrophila HA-4, a newly isolated cold-adapted sulfamethoxazole-degrading bacterium

Sulfamethoxazole is a common antibiotic that is frequently detected in wastewater and surface water. This study investigated the biodegradation and metabolic pathway of sulfamethoxazole by Pseudomonas psychrophila HA-4, a cold-adapted bacterium. Strain HA-4, which uses sulfamethoxazole as its sole source of carbon and energy, was isolated at a low temperature (10 °C) and identified as P. psychrophila by physico-biochemical characterization and 16S rRNA gene sequence analysis. Strain HA-4 removed sulfamethoxazole at temperatures ranging from 5.0 °C to 30 °C, with the maximal removal rate at 10 °C. The maximal removal rate of sulfamethoxazole by strain HA-4 was 34.30 % after 192 h at 10 °C. The highest percentage of unsaturated fatty acid was determined to be 23.03 % at 10 °C, which adheres to the characteristic for cold-adapted psychrophiles and psychrotrophs. At low concentrations of sulfamethoxazole, the growth kinetics correlated well with the Haldane model. The single-substrate parameter values of sulfamethoxazole on cell growth were determined to be μ _max?=?0.01 h^?1, K _s?=?20.91 mg/l and K _i?=?170.60 mg/l. Additionally, the major intermediates from sulfamethoxazole biodegradation by strain HA-4, including aniline, 3-amino-5-methylisoxazole, 4-aminothiophenol and sulfanilamide, were identified by GC-MS and high-resolution mass spectrometry (HR-MS) analysis. The results demonstrate that strain HA-4 has the potential to degrade sulfamethoxazole at low temperatures.     

Genetic variants in the JAK1 gene confer higher risk of Behcet’s disease with ocular involvement in Han Chinese

Recent surveys have identified SLC22A4, SLC22A5, RUNX1, JAK1 as susceptibility genes for various immune-related diseases. An association study was performed in 738 Behcet’s patients with ocular involvement and 1,873 controls using the iPLEX system method. The first-stage study for 30 SNPs showed that SNPs rs2780815, rs310241, rs3790532 in JAK1 were associated with Behcet’s disease in Han Chinese (Pc_{(Bonferroni correction)} = 0.022–7.7 × 10^?3). The G allele and AA genotype of SNP rs2834643 in RUNX1 (Pc = 0.041–1.75 × 10^?3), but none of the other SNPs, were associated with Behcet’s disease. Haplotype analysis for the SLC22A4, SLC22A5 genes showed an increased tendency for AGTCTGCCGC frequency in patients compared with controls; however, the significance was lost after Bonferroni correction (P = 0.004, Pc > 0.05). Subsequently, we further replicated the significantly associated SNPs using another independent cohort. Replication and combining studies showed that three SNPs rs2780815, rs310241, rs3790532 in JAK1, but not SNP rs2834643 in RUNX1, were consistently associated with Behcet’s disease (replication: Pc = 0.012–9.60 × 10^?4; combining: Pc = 0.030–1.90 × 10^?4). SNPs rs2780815, rs310241, rs3790532 were estimated to confer a population attributable risk of 35.0, 28.0, 27.0 %, respectively. We found a strong association between HLA-B51 with Behcet’s disease in Chinese Han population (P = 1.35 × 10^?73; OR = 5.15; 95 % CI 4.28–6.19). GMDR analysis showed that no gene–gene interaction was detectable between JAK1 and HLA-B51. Logistic analysis indicated that the JAK1 gene was an independent risk factor for Behcet’s disease (P > 0.05). Real-time PCR analysis showed that no difference on the expression of JAK1 in PBMCs or LPS-stimulated PBMCs between individuals with the different rs1762780815 genotypes studied (P > 0.05). In conclusion, this study suggests that JAK1, but not SLC22A4, SLC22A5 and RUNX1, contributes to the genetic susceptibility to Behcet’s disease with ocular involvement.     

Two-Probe Microfluorometry Estimation of Transmembrane Potential (ΔΨ p) Slight Changes in Individual Hepatocytes Under Short-Term Hormone Action

Early events in individual hepatocytes of rat activated by adrenaline (10^?6M) and phenylephrine (10^?5M) have been investigated by quantitative image microfluorometry and microspectrofluorometry. Cationic DiOC₂ and anionic SqSC₄ probes have been used for image analysis and transmembrane potential (ΔΨ _p) estimation in real-time studies. Fluorescence spectra resulting from the accumulation of dyes in single cells were recorded. Based on the mean fluorescence intensity, the magnitude of ΔΨ _p was calculated by Nernst equation adapted for lipophilic cationic probes. DiOC₂ has revealed that both hormones induce biphasic hyperpolarization of hepatocytes membrane with α-agonist phenylephrine causing ΔΨ _p changes at higher amplitude. The first increase of ΔΨ _p within 2 and 5 min (ΔΔΨ _p = ?8.6 ± 4.2 mV) apparently related to Na⁺/K⁺-ATPase activation by the Ca²⁺-mobilizing hormone. The second peak of hyperpolarization (ΔΔΨ _p = ?13.2 ± 3.2 mV) between 25 and 30 min, after a transient decrease of ΔΨ _p (ΔΔΨ _p = 10.9 ± 4.3 mV) over 15 min experiment, probably is mediated by phenylephrine stimulating action on K⁺-channels. K⁺ channel blocker (Ba²⁺ or 4-aminopyridine) as well as elevating of extracellular K⁺ prevented the hyperpolarization. Modulation of PLD-dependent signal transduction pathway by 0.4 % butanol had a weak influence on the first increase of ΔΨ _p but it abolished the second phase of hyperpolarization. That points to PLD involvement in the ΔΨ _p fluctuations mediated by K⁺-channels in response to phenylephrine. Based on SqSC₄, fluorescent parameters estimation of relative changes of ΔΨ _p revealed similar character of time dependence with two phases of hyperpolarization. Synchronic fluctuation of ΔΨ _p determined by oppositely charged probes demonstrate that the quantitative microfluorometry allows to evaluate slight ΔΨ _p changes separately from ΔΨ _m in non-excitable individual cells at the short-term hormone action.     

Induced leaf intercellular CO2, photosynthesis, potassium and nitrate retention and strawberry early fruit formation under macronutrient limitation

Relationships between induced high leaf intercellular CO₂ concentrations, leaf K⁺ and NO₃ ^? ion movement and early fruit formation under macronutrient limitation are not well understood. We examined the effects and interactions of reduced K/N input treatments on leaf intercellular CO₂, photosynthesis rate, carboxylation and water use efficiency, berry formation as well as leaf/fruit K⁺, NO₃ ^? and photosynthate retention of strawberry (Fragaria × ananassa Duch.) to enhance low-input agriculture. The field study was conducted in Nova Scotia, eastern Canada during 2009–2010. The experimental treatments consisted of five K₂O rates (0, 6, 12, 18, and 24 kg ha^?1) and five N rates (0, 5, 10, 15, and 20 kg ha^?1), representing respectively, 0, 25, 50, 75, and 100 % of regular macronutrient recommendations based on the soil testing. The treatments were arranged in a split-plot design with three blocks in the field. The cultivar was ‘Mira’, a June-bearing crop. The results showed that strawberry plants treated with 25 %-reduced inputs could induce significantly higher leaf intercellular CO₂ concentrations to improve plant photosynthesis, carboxylation and water use efficiency and translocation of leaf/fruit K⁺ and dissolved solids, which could advance berry formation by 6 days and produce significantly higher marketable yields (P < 0.05). Higher leaf intercellular CO₂ inhibited leaf/fruit NO₃ ^? ion retention, but this inhibition did not occur in leaf/fruit K⁺ retention. Linear interactions of the K/N treatments were significant on fruit marketable yields, intercellular CO₂, net photosynthesis, leaf transpiration rates, and leaf temperatures (P < 0.05). It was concluded that higher leaf CO₂ could enhance plant photosynthesis, promote plant carboxylation and water use efficiency, and advance berry formation, but it could inhibit leaf NO₃ ^? retention. This inhibition did not find in leaf K⁺ ion and dissolved solid retention. Overlay co-limitation of leaf intercellular CO₂ and translocation of leaf/fruit K⁺/NO₃ ^? and total dissolved solids could constrain more fruit formation attributes under full macronutrient supply than reduced inputs. It was suggested that low input would be an optimal and sustainable option for improving small fruit crop physiological development and dealing with macronutrient deficiency challenge.     

Saccharopolyspora dendranthemae sp. nov. a halotolerant endophytic actinomycete isolated from a coastal salt marsh plant in Jiangsu, China

A halotolerant actinomycete strain, designated strain KLBMP 1305^T, was isolated from a salt marsh plant Dendranthema indicum (Linn.) Des Moul collected from the coastal region of Nantong, Jiangsu Province, in east China and was studied in detail for its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain KLBMP 1305^T is a member of the genus Saccharopolyspora. The 16S rRNA gene sequence similarity indicated that strain KLBMP 1305^T was most closely related to ‘Saccharopolyspora pathumthaniensis’ S582^T (99.31 %), ‘Saccharopolyspora endophytica’ YIM 61095^T (99.17 %) and Saccharopolyspora tripterygii YIM 65359^T (99.15 %); similarity to other type strains of the genus Saccharopolyspora was <97.2 %. The organism had chemical and morphological features consistent with its classification in the genus Saccharopolyspora such as meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan and arabinose and galactose as the diagnostic sugars. The predominant menaquinone was MK-9(H₄). The polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unknown glycolipid and an unknown lipid. The major fatty acids were iso-C_16:0, iso-C_15:0, anteiso-C_15:0, anteiso-C_17:0 and sum in feature 8 (18:1ω7c/18:1ω6c)_. The G+C content of the genomic DNA of the type strain was 68.7 mol%. DNA–DNA relatedness data, together with phenotypic differences, clearly distinguished the isolate from its closest relatives. On the basis of these phenotypic and genotypic data, the isolate represents a novel species, for which the name Saccharopolyspora dendranthemae sp. nov. is proposed. The type strain is KLBMP 1305^T (=KCTC 19889^T = NBRC 108675^T).     

Transport and utilization of methionine sulfoxide in the rabbit

Methionine sulfoxide is transported into purified intestinal and renal brush border membrane vesicles from rabbit by an Na⁺-dependent mechanism and is accumulated inside the vesicles against the concentration gradient. Both in intestine and kidney, the rate of transport is enhanced with increasing concentrations of Na⁺ in the external medium. Increasing the Na⁺ gradient reduces the apparent K_t for methionine sulfoxide without causing any change in V_max. With an outward K⁺ gradient (vesicle > medium), valinomycin stimulates the Na⁺-gradient-dependent transport of methionine sulfoxide in the kidney, showing the electrogenicity of the transport process. A number of amino acids inhibit methionine sulfoxide transport in both the intestine and kidney. An enzymatic activity capable of reducing methionine sulfoxide to methionine is present in the intestinal mucosa, renal cortex and liver. The activity is highest in renal cortex and lowest in intestine. The methionine sulfoxide-reducing activity is stimulated by NADH, NADPH, glutathione and dithiothreitol and the potency of the stimulation is in the order: dithiothreitol > NADPH > glutathione > NADH.