Carbon,nitrogen and O2 fluxes associated with the cyanobacterium Nodularia spumigena in the Baltic Sea

Photosynthesis, respiration, N₂ fixation and ammonium release were studied directly in Nodularia spumigena during a bloom in the Baltic Sea using a combination of microsensors, stable isotope tracer experiments combined with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorometry. Cell-specific net C- and N₂-fixation rates by N. spumigena were 81.6±6.7 and 11.4±0.9 fmol N per cell per h, respectively. During light, the net C:N fixation ratio was 8.0±0.8. During darkness, carbon fixation was not detectable, but N₂ fixation was 5.4±0.4 fmol N per cell per h. Net photosynthesis varied between 0.34 and 250 nmol O₂ h⁻¹ in colonies with diameters ranging between 0.13 and 5.0 mm, and it reached the theoretical upper limit set by diffusion of dissolved inorganic carbon to colonies (>1 mm). Dark respiration of the same colonies varied between 0.038 and 87 nmol O₂ h⁻¹, and it reached the limit set by O₂ diffusion from the surrounding water to colonies (>1 mm). N₂ fixation associated with N. spumigena colonies (>1 mm) comprised on average 18% of the total N₂ fixation in the bulk water. Net NH₄⁺ release in colonies equaled 8–33% of the estimated gross N₂ fixation during photosynthesis. NH₄⁺ concentrations within light-exposed colonies, modeled from measured net NH₄⁺ release rates, were 60-fold higher than that of the bulk. Hence, N. spumigena colonies comprise highly productive microenvironments and an attractive NH₄⁺ microenvironment to be utilized by other (micro)organisms in the Baltic Sea where dissolved inorganic nitrogen is limiting growth.     

Hyaluronan Synthase 1 (HAS1) Requires Higher Cellular UDP-GlcNAc Concentration than HAS2 and HAS3

Mammals have three homologous genes encoding proteins with hyaluronan synthase activity (Has1–3), all producing an identical polymer from UDP-N-acetylglucosamine and UDP-glucuronic acid. To compare the properties of these isoenzymes, COS-1 cells, with minor endogenous hyaluronan synthesis, were transfected with human Has1–3 isoenzymes. HAS1 was almost unable to secrete hyaluronan or form a hyaluronan coat, in contrast to HAS2 and HAS3. This failure of HAS1 to synthesize hyaluronan was compensated by increasing the cellular content of UDP-N-acetyl glucosamine by ∼10-fold with 1 mm glucosamine in the growth medium. Hyaluronan synthesis driven by HAS2 was less affected by glucosamine addition, and HAS3 was not affected at all. Glucose-free medium, leading to depletion of the UDP-sugars, markedly reduced hyaluronan synthesis by all HAS isoenzymes while raising its concentration from 5 to 25 mm had a moderate stimulatory effect. The results indicate that HAS1 is almost inactive in cells with low UDP-sugar supply, HAS2 activity increases with UDP-sugars, and HAS3 produces hyaluronan at high speed even with minimum substrate content. Transfected Has2 and particularly Has3 consumed enough UDP-sugars to reduce their content in COS-1 cells. Comparison of different human cell types revealed ∼50-fold differences in the content of UDP-N-acetylhexosamines and UDP-glucuronic acid, correlating with the expression level of Has1, suggesting cellular coordination between Has1 expression and the content of UDP-sugars.     

Effect of N Source on the Steady State Growth and N Assimilation of P-limited Anabaena flos-aquae

Phosphate-limited chemostat cultures were used to study cell growth and N assimilation in Anabaena flos-aquae under various N sources to determine the relative energetic costs associated with the assimilation of NH₃, NO₃⁻, or N₂. Expressed as a function of relative growth rate, steady state cellular P contents and PO₄ assimilation rates did not vary with N-source. However, N-source did alter the maximal PO₄-limited growth rate achieved by the cultures: the NO₃⁻ and N₂ cultures attained only 97 and 80%, respectively, of the maximal growth rate of the NH₃ grown cells. Cellular biomass and C contents did not vary with growth rate, but changed with N source. The NO₃⁻-grown cells were the smallest (627 ± 34 micromoles C · 10⁻⁹ cells), while NH₃-grown cells were largest (900 ± 44 micromoles C · 10⁻⁹ cells) and N₂-fixing cells were intermediate (726 ± 48 micromoles C · 10⁻⁹ cells) in size. In the NO₃⁻-and N₂-grown cultures, N content per cell was only 57 and 63%, respectively, of that in the NH₃-grown cells. Heterocysts were absent in NH₃-grown cultures but were present in both the N₂ and NO₃⁻ cultures. In the NO₃⁻-grown cultures C₂H₂ reduction was detected only at high growth rates, where it was estimated to account for a maximum of 6% of the N assimilated. In the N₂-fixing cultures the acetylene:N₂ ratio varied from 3.4:1 at lower growth rates to 3.0:1 at growth rates approaching maximal.

Compared with NH₃, the assimilation of NO₃⁻ and N₂ resulted either in a decrease in cellular C (NO₃⁻ and N₂ cultures) or in a lower maximal growth rate (N₂ culture only). The observed changes in cell C content were used to calculate the net cost (in electron pair equivalents) associated with growth on NO₃⁻ or N₂ compared with NH₃.

     

Light-Limited Growth Rate Modulates Nitrate Inhibition of Dinitrogen Fixation in the Marine Unicellular Cyanobacterium Crocosphaera watsonii

Biological N₂ fixation is the dominant supply of new nitrogen (N) to the oceans, but is often inhibited in the presence of fixed N sources such as nitrate (NO₃ ⁻). Anthropogenic fixed N inputs to the ocean are increasing, but their effect on marine N₂ fixation is uncertain. Thus, global estimates of new oceanic N depend on a fundamental understanding of factors that modulate N source preferences by N₂-fixing cyanobacteria. We examined the unicellular diazotroph Crocosphaera watsonii (strain WH0003) to determine how the light-limited growth rate influences the inhibitory effects of fixed N on N₂ fixation. When growth (µ) was limited by low light (µ = 0.23 d⁻¹), short-term experiments indicated that 0.4 µM NH₄ ⁺ reduced N₂-fixation by ∼90% relative to controls without added NH₄ ⁺. In fast-growing, high-light-acclimated cultures (µ = 0.68 d⁻¹), 2.0 µM NH₄ ⁺ was needed to achieve the same effect. In long-term exposures to NO₃ ⁻, inhibition of N₂ fixation also varied with growth rate. In high-light-acclimated, fast-growing cultures, NO₃ ⁻ did not inhibit N₂-fixation rates in comparison with cultures growing on N₂ alone. Instead NO₃ ⁻ supported even faster growth, indicating that the cellular assimilation rate of N₂ alone (i.e. dinitrogen reduction) could not support the light-specific maximum growth rate of Crocosphaera. When growth was severely light-limited, NO₃ ⁻ did not support faster growth rates but instead inhibited N₂-fixation rates by 55% relative to controls. These data rest on the basic tenet that light energy is the driver of photoautotrophic growth while various nutrient substrates serve as supports. Our findings provide a novel conceptual framework to examine interactions between N source preferences and predict degrees of inhibition of N₂ fixation by fixed N sources based on the growth rate as controlled by light.     

Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances

The intramolecular distribution of nitrogen isotopes in N₂O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N₂O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N₂O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 ± 1.2‰, 32.5 ± 0.6‰, and 35.6 ± 1.4‰ for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N₂O from ammonia oxidation by N. europaea (31.4 ± 4.2‰) was similar to that produced during hydroxylamine oxidation (33.5 ± 1.2‰) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 ± 1.7‰), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N₂O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (−0.6 ± 1.9‰ and −0.5 ± 1.9‰, respectively) were similar to those during nitrate reduction (−0.5 ± 1.9‰ and −0.5 ± 0.6‰, respectively), indicating no influence of either substrate on site preference. Site preferences of ~33‰ and ~0‰ are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N₂O.     

Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production

An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar_At) has been characterized. βcar_At is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn²⁺, Ni²⁺, and Co²⁺. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcar_At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar_At is able to produce monosubstituted β²- and β³-amino acids, showing better catalytic efficiency (k_cat/K_m) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcar_At an outstanding candidate for application in the biotechnology industry.     

N2-fixation,ammonium release and N-transfer to the microbial and classical food web within a plankton community

We investigated the role of N₂-fixation by the colony-forming cyanobacterium, Aphanizomenon spp., for the plankton community and N-budget of the N-limited Baltic Sea during summer by using stable isotope tracers combined with novel secondary ion mass spectrometry, conventional mass spectrometry and nutrient analysis. When incubated with ¹⁵N₂, Aphanizomenon spp. showed a strong ¹⁵N-enrichment implying substantial ¹⁵N₂-fixation. Intriguingly, Aphanizomenon did not assimilate tracers of ¹⁵NH₄⁺ from the surrounding water. These findings are in line with model calculations that confirmed a negligible N-source by diffusion-limited NH₄⁺ fluxes to Aphanizomenon colonies at low bulk concentrations (<250 nm) as compared with N₂-fixation within colonies. No N₂-fixation was detected in autotrophic microorganisms <5 μm, which relied on NH₄⁺ uptake from the surrounding water. Aphanizomenon released about 50% of its newly fixed N₂ as NH₄⁺. However, NH₄⁺ did not accumulate in the water but was transferred to heterotrophic and autotrophic microorganisms as well as to diatoms (Chaetoceros sp.) and copepods with a turnover time of ~5 h. We provide direct quantitative evidence that colony-forming Aphanizomenon releases about half of its recently fixed N₂ as NH₄⁺, which is transferred to the prokaryotic and eukaryotic plankton forming the basis of the food web in the plankton community. Transfer of newly fixed nitrogen to diatoms and copepods furthermore implies a fast export to shallow sediments via fast-sinking fecal pellets and aggregates. Hence, N₂-fixing colony-forming cyanobacteria can have profound impact on ecosystem productivity and biogeochemical processes at shorter time scales (hours to days) than previously thought.     

Angiotensin II Stimulates Thick Ascending Limb Superoxide Production via Protein Kinase Cα-dependent NADPH Oxidase Activation

Angiotensin II (Ang II) stimulates thick ascending limb (TAL) O production, but the receptor(s) and signaling mechanism(s) involved are unknown. The effect of Ang II on O is generally attributed to the AT₁ receptor. In some cells, Ang II stimulates protein kinase C (PKC), whose α isoform (PKCα) can activate NADPH oxidase. We hypothesized that in TALs, Ang II stimulates O via AT₁ and PKCα-dependent NADPH oxidase activation. In rat TALs, 1 nm Ang II stimulated O from 0.76 ± 0.17 to 1.97 ± 0.21 nmol/min/mg (p < 0.001). An AT₁ antagonist blocked the stimulatory effect of Ang II on O (0.87 ± 0.25 nmol/min/mg; p < 0.006), whereas an AT₂ antagonist had no effect (2.16 ± 0.133 nmol/min/mg; p < 0.05 versus vehicle). Apocynin, an NADPH oxidase inhibitor, blocked Ang II-stimulated O by 90% (p < 0.01). Ang II failed to stimulate O in TALs from p47^phox−/− mice (p < 0.02). Monitored by fluorescence resonance energy transfer, Ang II increased PKC activity from 0.02 ± 0.03 to 0.13 ± 0.02 arbitrary units (p < 0.03). A general PKC inhibitor, GF109203X, blocked the effect of Ang II on O (1.47 ± 0.21 versus 2.72 ± 0.47 nmol/min/mg with Ang II alone; p < 0.03). A PKCα- and β-selective inhibitor, Gö6976, also blocked the stimulatory effect of Ang II on O (0.59 ± 0.15 versus 2.05 ± 0.28 nmol/min/mg with Ang II alone; p < 0.001). To distinguish between PKCα and PKCβ, we used tubules expressing dominant-negative PKCα or -β. In control TALs, Ang II stimulated O by 2.17 ± 0.44 nmol/min/mg (p < 0.011). In tubules expressing dominant-negative PKCα, Ang II failed to stimulate O (change: −0.30 ± 0.27 nmol/min/mg). In tubules expressing dominant-negative PKCβ1, Ang II stimulated O by 2.08 ± 0.69 nmol/min/mg (p < 0.002). We conclude that Ang II stimulates TAL O production via activation of AT₁ receptors and PKCα-dependent NADPH oxidase.     

Perfusion Method for Assaying Microbial Activities in Sediments: Applicability to Studies of N2 Fixation by C2H2 Reduction

A perfusion method for assaying nitrogenase activity (acetylene reduction) in marine sediments was developed. The method was used to assay sediment cores from Spartina alterniflora (salt marsh), Zostera marina (sea grass), and Thalassia testudinum (sea grass) communities, and the results were compared with those of conventional sealed-flask assays. Rates of ethylene production increased progressively with time in the perfusion assays, reaching plateau values of 2 to 3 nmol · g of dry sediment⁻¹ · h⁻¹ by 10 to 20 h. Depletion of interstitial NH₄⁺ was implicated in this stimulation of nitrogenase activity. Initial acetylene reduction rates determined by the perfusion assay of cores from the Spartina community ranged from 0.15 to 0.60 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹. These rates were similar to those for sediments assayed in sealed flasks without seawater when determined over linear periods of C₂H₄ production. Initial values obtained by using the perfusion method were 0.66 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Zostera communities and 0.70 nmol of C₂H₄ · g of dry sediment⁻¹ · h⁻¹ for sediments from Thalassia communities. In all cases, rates determined by simultaneous slurry assays were lower than those determined by the perfusion method.     

Purification and Characterization of the Coniferyl Aldehyde Dehydrogenase from Pseudomonas sp. Strain HR199 and Molecular Characterization of the Gene

The coniferyl aldehyde dehydrogenase (CALDH) of Pseudomonas sp. strain HR199 (DSM7063), which catalyzes the NAD⁺-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 ± 5,000 Da, and the subunit mass was 49.5 ± 2.5 kDa, indicating an α₂ structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26°C. The K_m values for coniferyl aldehyde and NAD⁺ were about 7 to 12 μM and 334 μM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH₂-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (calB). The corresponding nucleotide sequence was localized on a 9.4-kbp EcoRI fragment (E94), which was subcloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD⁺-dependent aldehyde dehydrogenases from different sources.     

High rates of denitrification and nitrous oxide emission in arid biological soil crusts from the Sultanate of Oman

Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Complete denitrification to N₂ was further confirmed by an ¹⁵NO₃⁻ tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Strikingly, N₂O gas was emitted at very high potential rates of 387±143 and 31±6 μmol N m⁻² h⁻¹ from the cyanobacterial and lichen crust, respectively, with N₂O accounting for 53–66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N₂O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N₂O gas emission and potentially reduces desert soil fertility.     

Methyl-β-cyclodextrin Suppresses Hyaluronan Synthesis by Down-regulation of Hyaluronan Synthase 2 through Inhibition of Akt

Hyaluronan synthases (HAS1–3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-β-cyclodextrin (MβCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MβCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MβCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MβCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MβCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.     

Functional size of photosynthetic electron transport chain determined by radiation inactivation

Radiation inactivation technique was employed to determine the functional size of photosynthetic electron transport chain of spinach chloroplasts. The functional size for photosystem I+II (H₂O to methylviologen) was 623 ± 37 kilodaltons; for photosystem II (H₂O to dimethylquinone/ferricyanide), 174 ± 11 kilodaltons; and for photosystem I (reduced diaminodurene to methylviologen), 190 ± 11 kilodaltons. The difference between 364 ± 22 (the sum of 174 ± 11 and 190 ± 11) kilodaltons and 623 ± 37 kilodaltons is partially explained to be due to the presence of two molecules of cytochrome b₆/f complex of 280 kilodaltons. The molecular mass for other partial reactions of photosynthetic electron flow, also measured by radiation inactivation, is reported. The molecular mass obtained by this technique is compared with that determined by other conventional biochemical methods. A working hypothesis for the composition, stoichiometry, and organization of polypeptides for photosynthetic electron transport chain is proposed.     

Maize Yield Response to Water Supply and Fertilizer Input in a Semi-Arid Environment of Northeast China

Maize grain yield varies highly with water availability as well as with fertilization and relevant agricultural management practices. With a 311-A optimized saturation design, field experiments were conducted between 2006 and 2009 to examine the yield response of spring maize (Zhengdan 958, Zea mays L) to irrigation (I), nitrogen fertilization (total nitrogen, urea-46% nitrogen,) and phosphorus fertilization (P₂O₅, calcium superphosphate-13% P₂O₅) in a semi-arid area environment of Northeast China. According to our estimated yield function, the results showed that N is the dominant factor in determining maize grain yield followed by I, while P plays a relatively minor role. The strength of interaction effects among I, N and P on maize grain yield follows the sequence N+I >P+I>N+P. Individually, the interaction effects of N+I and N+P on maize grain yield are positive, whereas that of P+I is negative. To achieve maximum grain yield (10506.0 kg·ha⁻¹) for spring maize in the study area, the optimum application rates of I, N and P are 930.4 m³·ha⁻¹, 304.9 kg·ha⁻¹ and 133.2 kg·ha⁻¹ respectively that leads to a possible economic profit (EP) of 10548.4 CNY·ha⁻¹ (CNY, Chinese Yuan). Alternately, to obtain the best EP (10827.3 CNY·ha⁻¹), the optimum application rates of I, N and P are 682.4 m³·ha⁻¹, 241.0 kg·ha⁻¹ and 111.7 kg·ha⁻¹ respectively that produces a potential grain yield of 10289.5 kg·ha⁻¹.     

Emissions of NO and NH3 from a Typical Vegetable-Land Soil after the Application of Chemical N Fertilizers in the Pearl River Delta

Cropland soil is an important source of atmospheric nitric oxide (NO) and ammonia (NH₃). Chinese croplands are characterized by intensive management, but limited information is available with regard to NO emissions from croplands in China and NH₃ emissions in south China. In this study, a mesocosm experiment was conducted to measure NO and NH₃ emissions from a typical vegetable-land soil in the Pearl River Delta following the applications of 150 kg N ha⁻¹ as urea, ammonium nitrate (AN) and ammonium bicarbonate (ABC), respectively. Over the sampling period after fertilization (72 days for NO and 39 days for NH₃), mean NO fluxes (± standard error of three replicates) in the control and urea, AN and ABC fertilized mesocosms were 10.9±0.9, 73.1±2.9, 63.9±1.8 and 66.0±4.0 ng N m⁻² s⁻¹, respectively; mean NH₃ fluxes were 8.9±0.2, 493.6±4.4, 144.8±0.1 and 684.7±8.4 ng N m⁻² s⁻¹, respectively. The fertilizer-induced NO emission factors for urea, AN and ABC were 2.6±0.1%, 2.2±0.1% and 2.3±0.2%, respectively. The fertilizer-induced NH₃ emission factors for the three fertilizers were 10.9±0.2%, 3.1±0.1% and 15.2±0.4%, respectively. From the perspective of air quality protection, it would be better to increase the proportion of AN application due to its lower emission factors for both NO and NH₃.     

Nitrate-Dependent Ferrous Iron Oxidation by Anaerobic Ammonium Oxidation (Anammox) Bacteria

We examined nitrate-dependent Fe²⁺ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe²⁺ and reduced NO₃⁻ to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein⁻¹ min⁻¹, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca. Brocadia sinica” (10 to 75 nmol NH₄⁺ mg protein⁻¹ min⁻¹). A ¹⁵N tracer experiment demonstrated that coupling of nitrate-dependent Fe²⁺ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO₃⁻ by “Ca. Brocadia sinica.” The activities of nitrate-dependent Fe²⁺ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO₃⁻ ± SD of “Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe²⁺ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe²⁺ oxidation and NO₃⁻ reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein⁻¹ min⁻¹, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe²⁺ oxidation and the anammox reaction decreased the molar ratios of consumed NO₂⁻ to consumed NH₄⁺ (ΔNO₂⁻/ΔNH₄⁺) and produced NO₃⁻ to consumed NH₄⁺ (ΔNO₃⁻/ΔNH₄⁺). These reactions are preferable to the application of anammox processes for wastewater treatment.     

Transporting Antitumor Drug Tamoxifen and Its Metabolites, 4-Hydroxytamoxifen and Endoxifen by Chitosan Nanoparticles

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K _tam-ch-15  = 8.7 (±0.5)×10³ M⁻¹, K _tam-ch-100  = 5.9 (±0.4)×10⁵ M⁻¹, K _tam-ch-200  = 2.4 (±0.4)×10⁵ M⁻¹ and K _{hydroxytam-ch-15}  = 2.6(±0.3)×10⁴ M⁻¹, K _{hydroxytam – ch-100}  = 5.2 (±0.7)×10⁶ M⁻¹ and K _{hydroxytam-ch-200}  = 5.1 (±0.5)×10⁵ M⁻¹, K _endox-ch-15  = 4.1 (±0.4)×10³ M⁻¹, K _endox-ch-100  = 1.2 (±0.3)×10⁶ M⁻¹ and K _endox-ch-200  = 4.7 (±0.5)×10⁵ M⁻¹ with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH₂ residues with drug OH and NH₂ groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.     

Zinc flexes its muscle: Correcting a novel analysis of calcium for zinc interference uncovers a method to measure zinc

The divalent cation chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), often used to buffer physiological changes in cytosolic Ca²⁺, also binds Zn²⁺ with high affinity. In a recently published method (Lamboley et al. 2015. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201411250), the absorbance shift of BAPTA at 292 nm was successfully used to determine the total calcium concentrations of various skeletal muscle tissues. In the present study, we show that endogenous Zn²⁺ in rat skeletal muscle tissue can be unknowingly measured as “Ca²⁺,” unless appropriate measures are taken to eliminate Zn²⁺ interference. We analyzed two rat skeletal muscle tissues, soleus and plantaris, for total calcium and zinc using either inductively coupled plasma mass spectrometry (ICP-MS) or the BAPTA method described above. ICP-MS analysis showed that total zinc contents in soleus and plantaris were large enough to affect the determination of total calcium by the BAPTA method (calcium = 1.72 ± 0.31 and 1.96 ± 0.14, and zinc = 0.528 ± 0.04 and 0.192 ± 0.01; mean ± standard error of the mean [SEM]; n = 5; mmole/kg, respectively). We next analyzed total calcium using BAPTA but included the Zn²⁺-specific chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) that buffers Zn²⁺ without affecting Ca²⁺/BAPTA binding. We found that estimated concentrations of total calcium ([Ca_T]_WM) in soleus and plantaris were reduced after TPEN addition ([Ca_T]_WM = 3.71 ± 0.62 and 3.57 ± 0.64 without TPEN and 3.39 ± 0.64 and 3.42 ± 0.62 with TPEN; mean ± SEM; n = 3; mmole/kg, respectively). Thus, we show that a straightforward correction can be applied to the BAPTA method to improve the accuracy of the determination of total calcium that should be applicable to most any tissue studied. In addition, we show that using TPEN in combination with the BAPTA method allows one to make reasonable estimates of total zinc concentration that are in agreement with the direct determination of zinc concentration by ICP-MS.