Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by ¹⁵N₂ labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography-mass spectrometry. In detached nodules of Myrica , glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ-amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse-chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1-h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.     

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

The calcineurin activity and MCIP1.4 mRNA levels are increased by innervation in regenerating soleus muscle

The level of active subunit of calcineurin and the calcineurin (Cn) enzyme activity are increased in innervated but not in denervated slow type regenerating skeletal soleus muscle. These nerve-dependent increases were not accompanied by similar increases in the mRNA levels. The changes in the mRNA level of the modulatory calcineurin interacting protein, MCIP1.4, reflected the calcineurin activity and did not increase in denervated regenerating muscles compared to the innervated regenerating controls. The increases in Cn activity and in MCIP1.4 mRNA levels occurred before the switch from fast to slow-type myosin heavy chain isoforms, a phenomenon similarly known to be dependent on innervation. This highlights the role of mediators, acting between the nerve and calcineurin, in the formation of slow fiber identity.     

Non-nodulating pink-pigmented facultative Methylobacterium sp. with a functional nifH gene

Among the 250 Methylobacterium isolates studied, 11 were able to grow in Nitrogen-free methanol mineral salts medium. Out of the eleven isolates, except MV10, 10 isolates had the nodA gene. ARA and presence of nifH gene confirmed the ability of isolate MV10 to fix biological nitrogen. Plant infection tests conducted with Crotalaria sp. confirmed the inability of isolate MV10 to nodulate Crotalaria sp. The presence of a functional nifH gene and absence of a nodA gene differentiate this isolate from the other 15 species so far described in the genus Methylobacterium and suggest that it is a new species.     

Genomic data for alternate production strategies. I. Identification of major contaminating species for Cobalt(+2) immobilized metal affinity chromatography

Recent advances in technology have allowed for the identification of complex protein mixtures in a rapid fashion. This report highlights the use of 2D gel electrophoresis, mass spectrometry, and database analysis to determine contaminating species of the Escherichia coli genome that are present during immobilized metal affinity chromatography (IMAC), highlighting Co(2+) as the affinity ligand. Four proteins (triosephosphate isomerase, alpha galactosidase, Hsp90, and glucosamine 6-phosphate synthase) constitute the majority of E. coli proteins that bind and potentially may coelute during chromatography. Results are discussed within the context of changes that when implemented could lead to an increase in IMAC efficiency, not by altering column conditions, but rather by changing the nature of the nuisance proteins that principally reduce column capacity and extend processing times. Such a study illustrates the use of proteome data to aid in bioprocess design.     

Effects of photoperiodicity and light irradiance on phosphate-limited Oscillatoria agardhii in chemostat cultures

Oscillatoria sp. strain 23 is a filamentous, non-heterocystous cyanobacterium that fixes nitrogen aerobically. Although, in this organism nitrogenase is inactivated by oxygen a high tolerance is observed. Up to a pO₂ of 0.15 atm, oxygen does not have any measurable effects on acetylene reduction. Higher concentrations of oxygen inhibited the activity to a relatively high degree. Evidence for two mechanisms of oxygen protection of nitrogenase in this cyanobacterium was obtained. A high rate of synthesis of nitrogenase may allow the organism to maintain a certain amount of active enzyme under aerobic conditions. Secondly, a switch off/on mechanism may reversibly convert the active enzyme into a non-active form which is insensitive to oxygen inactivation after a sudden and short-term exposure to high oxygen concentrations. It is conceived that these mechanisms in addition to a temporal separation of nitrogen fixation from oxygenic photosynthesis sufficiently explain the regulation process of aerobic nitrogen fixation in this organism.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - CAP chloramphenicol     

表达条件对金属激活酶NAOase离子依赖的活性分析

N-乙酰鸟氨酸脱酰基酶为一种新型手性拆分酶制剂,酶活性依赖于Mg2＋、Mn2＋、Zn2＋及Co2＋ 中的某种金属离子。以高表达NAOase的重组菌DH10B/argE-pHsh为研究对象,考察不同培养条件下Mg2＋、Mn2＋、Zn2＋及Co2＋ 4种离子对重组菌的生长、酶的表达活性及表达量的影响。结果发现：同种离子在不同条件下对重组菌的生长影响不大,但对酶活性影响显著。4种离子在合适浓度时皆能提高酶活性,促进强度从高到低依次为Mn2＋、Mg2＋、Co2＋和Zn2＋。在TB培养基中,Mn2＋为15 mmol/L时：NAOase比酶活达到1 272.7 U/mL,是未添加时的4.67倍,激活作用显著高于其他离子。SDS-PAGE电泳实验表明4种离子的蛋白表达量基本相同。     

Physiological regulation of carbon fixation in the photosynthesis and calcification of coccolithophorids

Emiliania huxleyi and Gephyrocapsa oceanica are the predominant coccolithophorid species that produce blooms in the ocean and affect the global environment. These species are capable of carbon fixation by both photosynthesis for organic matter production and by intracellular calcification for coccolith production. Both processes were strongly affected by the nutrient status in a laboratory culture. The coccolith production was stimulated by the addition of a high concentration of sodium bicarbonate and by the depletion of phosphate. Interestingly, when the calcification was stimulated, the increase in cell number during algal growth was greatly suppressed and then the cell volume increased. When the growth rate was increased under nutrient-sufficient conditions, the cells became very small in size and most of them bore few or no coccoliths. The data from laboratory experiments show that the cell growth and calcification proceeded apparently independently at different phases. We, therefore, assume that the coccolithophorid blooms in the ocean might be separated into two phases; firstly, the increase in cell population might be triggered by an adequate supply of nutrients to enhance algal growth and then the calcification might subsequently be stimulated when the nutrients become depleted by substantial algal growth.     

Immobilization and phytoavailability of cadmium in variable charge soils. I. Effect of phosphate addition

The effect of phosphate on the surface charge and cadmium (Cd) adsorption was examined in seven soils that varied in their variable-charge components. The effect of phosphate on immobilization and phytoavailability of Cd from one of the soils, treated with various levels of Cd (0–10 mg Cd kg^–1 soil), was further evaluated using mustard (Brassica juncea L.) plants. Cadmium immobilization in soil was evaluated by a chemical fractionation scheme. Addition of phosphate, as KH₂PO₄, increased the pH, negative charge and Cd adsorption by the soils. Of the seven soils examined, the three allophanic soils (i.e., Egmont, Patua and Ramiha) exhibited greater increases in phosphate-induced pH, negative charge and Cd²⁺ adsorption over the other four non-allophanic soils (i.e., Ballantrae, Foxton, Manawatu ad Tokomaru). Increasing addition of Cd enhanced Cd concentration in plants, resulting in decreased plant growth (i.e., phytotoxicity). Addition of phosphate effectively reduced the phytotoxicity of Cd. There was a significant inverse relationship between dry matter yield and Cd concentration in soil solution. Addition of phosphate decreased the concentration of the soluble + exchangeable Cd fraction but increased the concentration of inorganic-bound Cd fraction in soil. The phosphate-induced alleviation of Cd phytotoxicity can be attributed primarily to Cd immobilization due to increases in pH and surface charge.     

Use of crops for in situ phytoremediation of polluted soils following a toxic flood from a mine spill

Following a toxic flood from a mine spill that affected over 45 km² in Southern Spain, experiments were conducted in 1999 to test the feasibility of using crops for phytoremediation of the area, after the mechanical removal of the mud. Two cereals, barley and triticale, and two Brassicaspp., rapeseed and ethiopian mustard, were planted in three contaminated plots, 50 × 100 m each, and in a control plot outside the affected area. Soil and plant contents of As, Cd, Cu, Pb, Tl and Zn were measured and bioaccumulation coefficients (BC) were calculated at maturity. The four crops tested accumulated Cd and Zn in the above-ground biomass only in the plot on acid soil. Both species of Brassica accumulated Tl (average BC of 3.6 and 1.4 for rapeseed and mustard, respectively, in contaminated plots). None of the four crop plants accumulated As, Cu and Pb under the experimental conditions. Maximum plant uptake values from soil were 5.4 mg m^–2 of As, 0.54 mg m^–2 of Cd, 9.7 mg m^–2 of Cu, 7.0 mg m^–2 of Pb, 3.4 mg m^–2 of Tl, and 260 mg m^–2 of Zn. Total crop uptake gave estimates for successful phytoremediation of at least five decades, casting doubts on the feasibility of using these crops for decontamination of the area. Nevertheless, cereal grains had mineral contents below toxicity levels for livestock, therefore it might be possible to use these crops for livestock feed while reducing deep percolation and gradually removing metals from polluted soils.