Ammonium uptake by Chlorella

The preincubation of Chlorella cells with glucose caused a tenfold increase of the maximal uptake rate of ammonium without change in the K _m (2 M). A similar stimulation of ammonium uptake was found when the cells were transferred to nitrogen-free growth medium. The time-course of uptake stimulation by glucose revealed a lag period of 10–20 min. The turnover of the ammonium transport system is characterized by a half-life time of 5–10 h, but in the presence of light 30% of uptake activity stayed even after 50 h. 6-Deoxyglucose was not able to increase the ammonium uptake rate. These data together were interpreted as evidence for induction of an ammonium transport system by a metabolite of glucose. Mechanistic studies of the ammonium transport system provided evidence for the electrogenic uptake of the ammonium ion. The charge compensation for NH ₄ ⁺ entry was achieved by immediate K⁺ efflux from the cells, and this was followed after 1 min by H⁺ extrusion. Ammonium accumulated in the cells; the rate of uptake was sensitive to p-trifluoromethoxy-carbonylcyanide-phenylhydrazon and insensitive to methionine-sulfoxime. Uptake studies with methylamine revealed that methylamine transport is obviously catalyzed by the ammonium transport system and, therefore, also increased in glucose-treated Chlorella cells.Abbreviation p.c. packed cells     

Alkylguanidines as inhibitors of k transport in isolated barley roots

It has been shown that plants can accumulate K⁺ through an energy-dependent process. The effect of alkylguanidines, in particular octylguanidine on the uptake of ⁸⁶Rb⁺ by excised barley roots (Hordeum vulgare var. Apizaco LV-72), has been studied. ⁸⁶Rb⁺ was used as tracer of K⁺. The uptake of ⁸⁶Rb⁺ which is linear with time and shows saturation kinetics is inhibited by octylguanidine. Half-maximal inhibition of ⁸⁶Rb⁺ uptake is attained at 50 μM octylguanidine. Octylguanidine induces a decrease in the V_max of the process and increases the Km of the system for Rb⁺. When the effects of various alkylguanidines were studied, the following order of effectiveness was encountered; octylguanidine = hexilguanidine > butylguanidine > ethylguanidine > guanidine. This suggests that guanidines inhibit Rb⁺ uptake by interacting through its positively charged guanidinium group with a Rb⁺ carrier while the alkyl chain interacts with the hydrophobic milieu of the membrane.     

Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO₃ ⁻), ammonium (NH₄ ⁺), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L⁻¹ for NO₃ ⁻ and NH₄ ⁺, 0.5–50 μmol N L⁻¹ for urea, 0.5–100 μmol N L⁻¹ for glu and cyanate, and 0.5–200 μmol N L⁻¹ for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μ_m, 1.51±0.06 d⁻¹ and 1.50±0.05 d⁻¹, respectively). However, cultures grown on cyanate, NO₃ ⁻, and NH₄ ⁺ had lower half saturation constants (K_μ, 0.28–0.51 μmol N L⁻¹). N uptake kinetics were measured in NO₃ ⁻-deplete and -replete batch cultures of P. donghaiense. In NO₃ ⁻-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO₃ ⁻, NH₄ ⁺, urea and algal amino acids; uptake was saturated at or below 50 μmol N L⁻¹. In NO₃ ⁻-replete batch cultures, NH₄ ⁺, urea, and algal amino acid uptake kinetics were similar to those measured in NO₃ ⁻-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.     

Characterization of nigericin-stimulated ATPase from sealed microsomal vesicles of tobacco callus

To understand the function and membrane origin of ionophore-stimulated ATPases, the activity of nigericin-stimulated ATPase was characterized from a low-density microsomal fraction containing sealed vesicles of autonomous tobacco (Nicotiana tabacum Linnaeous cv. Wisconsin no. 38) callus. The properties of KCl-stimulated, Mg-requiring ATPases (KCl-Mg,ATPase) were similar in the absence or presence of nigericin. Nigericin (or gramicidin) stimulation of a KCl-Mg,ATPase activity was optimum at pH 6.5 to 7.0. The enzyme was inhibited completely by N,N′-dicyclohexylcarbodiimide (10 μm), tributyltin (5 μm), and partially by vanadate (200 μm), but it was insensitive to fusicoccin and mitochondrial ATPase inhibitors, such as azide (1 mm) and oligomycin (5 μg/ml). The ATPase was more sensitive to anions than cations. Cations stimulated ATPase activity with a selectivity sequence of NH₄⁺ > K⁺, Rb⁺, Cs⁺, Na⁺, Li⁺ > Tris⁺. Anions stimulated Mg, ATPase activity with a decreasing sequence of Cl⁻ = acetate > SO₄²⁻ > benzene sulfonate > NO₃⁻. The anion stimulation was caused partly by dissipation of the electrical potential (interior positive) by permeant anions and partly by a specific ionic effect. Plant membranes had at least two classes of nigericin-stimulated ATPases: one sensitive and one insensitive to vanadate. Many of the properties of the nigericin-sensitive, salt-stimulated Mg,ATPase were similar to a vanadate-sensitive plasma membrane ATPase of plant tissues, yet other properties (anion stimulation and vanadate insensitivity) resembled those of a tonoplast ATPase. These results support the idea that nigericin-stimulated ATPases are mainly electrogenic H⁺ pumps originated in part from the plasma membrane and in part from other nonmitochondrial membranes, such as the tonoplast.     

Adenosine Triphosphate Activates a Noninactivating K+ Current in Adrenal Cortical Cells through Nonhydrolytic Binding

Bovine adrenal zona fasciculata (AZF) cells express a noninactivating K⁺ current (I_AC) that is inhibited by adrenocorticotropic hormone and angiotensin II at subnanomolar concentrations. Since I_AC appears to set the membrane potential of AZF cells, these channels may function critically in coupling peptide receptors to membrane depolarization, Ca²⁺ entry, and cortisol secretion. I_AC channel activity may be tightly linked to the metabolic state of the cell. In whole cell patch clamp recordings, MgATP applied intracellularly through the patch electrode at concentrations above 1 mM dramatically enhanced the expression of I_AC K⁺ current. The maximum I_AC current density varied from a low of 8.45 ± 2.74 pA/pF (n = 17) to a high of 109.2 ± 26.3 pA/pF (n = 6) at pipette MgATP concentrations of 0.1 and 10 mM, respectively. In the presence of 5 mM MgATP, I_AC K⁺ channels were tonically active over a wide range of membrane potentials, and voltage-dependent open probability increased by only ∼30% between −40 and +40 mV. ATP (5 mM) in the absence of Mg²⁺ and the nonhydrolyzable ATP analog AMP-PNP (5 mM) were also effective at enhancing the expression of I_AC, from a control value of 3.7 ± 0.1 pA/pF (n = 3) to maximum values of 48.5 ± 9.8 pA/pF (n = 11) and 67.3 ± 23.2 pA/pF (n = 6), respectively. At the single channel level, the unitary I_AC current amplitude did not vary with the ATP concentration or substitution with AMP-PNP. In addition to ATP and AMP-PNP, a number of other nucleotides including GTP, UTP, GDP, and UDP all increased the outwardly rectifying I_AC current with an apparent order of effectiveness: MgATP > ATP = AMP-PNP > GTP = UTP > ADP >> GDP > AMP and ATP-γ-S. Although ATP, GTP, and UTP all enhanced I_AC amplitude with similar effectiveness, inhibition of I_AC by ACTH (200 pM) occurred only in the presence of ATP. As little as 50 μM MgATP restored complete inhibition of I_AC, which had been activated by 5 mM UTP. Although the opening of I_AC channels may require only ATP binding, its inhibition by ACTH appears to involve a mechanism other than hydrolysis of this nucleotide. These findings describe a novel form of K⁺ channel modulation by which I_AC channels are activated through the nonhydrolytic binding of ATP. Because they are activated rather than inhibited by ATP binding, I_AC K⁺ channels may represent a distinctive new variety of K⁺ channel. The combined features of I_AC channels that allow it to sense and respond to changing ATP levels and to set the resting potential of AZF cells, suggest a mechanism where membrane potential, Ca²⁺ entry, and cortisol secretion could be tightly coupled to the metabolic state of the cell through the activity of I_AC K⁺ channels.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Anion-sensitive, h-pumping ATPase in membrane vesicles from oat roots

H⁺-pumping ATPases were detected in microsomal vesicles of oat (Avena sativa L. var Lang) roots using [¹⁴C]methylamine distribution or quinacrine fluorescent quenching. Methylamine (MeA) accumulation into vesicles and quinacrine quench were specifically dependent on Mg,ATP. Both activities reflected formation of a proton gradient (ΔpH) (acid inside) as carbonyl cyanide m-chlorophenylhydrazone, nigericin (in the presence of K⁺), or gramicidin decreased MeA uptake or increased quinacrine fluorescence. The properties of H⁺ pumping as measured by MeA uptake were characterized. The K_mapp for ATP was about 0.1 millimolar. Mg,GTP and Mg, pyrophosphate were 19% and 30% as effective as Mg,ATP. MeA uptake was inhibited by N,N′-dicyclohexylcarbodiimide and was mostly insensitive to oligomycin, vanadate, or copper. ATP-dependent MeA was stimulated by anions with decreasing order of potency of Cl⁻ > Br⁻ > NO₃⁻ > SO₄²⁻, iminodiacetate, benzene sulfonate. Anion stimulation of H⁺ pumping was caused in part by the ability of permeant anions to dissipate the electrical potential and in part by a specific requirement of Cl⁻ by a H⁺ -pumping ATPase. A pH gradient, probably caused by a Donnan potential, could be dissipated by K⁺ in the presence or absence of ATP. MeA uptake was enriched in vesicles of relatively low density and showed a parallel distribution with vanadate-insensitive ATPase activity on a continuous dextran gradient. ΔpH as measured by quinacrine quench was partially vanadate-sensitive. These results show that plant membranes have at least two types of H⁺ -pumping ATPases. One is vanadate-sensitive and probably enriched in the plasma membrane. One is vanadate-resistant, anion-sensitive and has many properties characteristic of a vacuolar ATPase. These results are consistent with the presence of electrogenic H⁺ pumps at the plasma membrane and tonoplast of higher plant cells.     

Different transport mechanisms in plant and human AMT/Rh-type ammonium transporters

The conserved family of AMT/Rh proteins facilitates ammonium transport across animal, plant, and microbial membranes. A bacterial homologue, AmtB, forms a channel-like structure and appears to function as an NH₃ gas channel. To evaluate the function of eukaryotic homologues, the human RhCG glycoprotein and the tomato plant ammonium transporter LeAMT1;2 were expressed and compared in Xenopus oocytes and yeast. RhCG mediated the electroneutral transport of methylammonium (MeA), which saturated with K_m = 3.8 mM at pH_o 7.5. Uptake was strongly favored by increasing the pH_o and was inhibited by ammonium. Ammonium induced rapid cytosolic alkalinization in RhCG-expressing oocytes. Additionally, RhCG expression was associated with an alkali-cation conductance, which was not significantly permeable to NH₄⁺ and was apparently uncoupled from the ammonium transport. In contrast, expression of the homologous LeAMT1;2 induced pH_o-independent MeA⁺ uptake and specific NH₄⁺ and MeA⁺ currents that were distinct from endogenous currents. The different mechanisms of transport, including the RhCG-associated alkali-cation conductance, were verified by heterologous expression in appropriate yeast strains. Thus, homologous AMT/Rh-type proteins function in a distinct manner; while LeAMT1;2 carries specifically NH₄⁺, or cotransports NH₃/H⁺, RhCG mediates electroneutral NH₃ transport.     

Certhrax Toxin,an Anthrax-related ADP-ribosyltransferase from Bacillus cereus

We identified Certhrax, the first anthrax-like mART toxin from the pathogenic G9241 strain of Bacillus cereus. Certhrax shares 31% sequence identity with anthrax lethal factor from Bacillus anthracis; however, we have shown that the toxicity of Certhrax resides in the mART domain, whereas anthrax uses a metalloprotease mechanism. Like anthrax lethal factor, Certhrax was found to require protective antigen for host cell entry. This two-domain enzyme was shown to be 60-fold more toxic to mammalian cells than anthrax lethal factor. Certhrax localizes to distinct regions within mouse RAW264.7 cells by 10 min postinfection and is extranuclear in its cellular location. Substitution of catalytic residues shows that the mART function is responsible for the toxicity, and it binds NAD⁺ with high affinity (K_D = 52.3 ± 12.2 μm). We report the 2.2 Å Certhrax structure, highlighting its structural similarities and differences with anthrax lethal factor. We also determined the crystal structures of two good inhibitors (P6 (K_D = 1.7 ± 0.2 μm, K_i = 1.8 ± 0.4 μm) and PJ34 (K_D = 5.8 ± 2.6 μm, K_i = 9.6 ± 0.3 μm)) in complex with Certhrax. As with other toxins in this family, the phosphate-nicotinamide loop moves toward the NAD⁺ binding site with bound inhibitor. These results indicate that Certhrax may be important in the pathogenesis of B. cereus.     

Kinetics of Sulfate and Acetate Uptake by Desulfobacter postgatei

The kinetics of sulfate and acetate uptake was studied in the sulfate-reducing bacterium Desulfobacter postgatei (DSM 2034). Kinetic parameters (K_m and V_max) were estimated from substrate consumption curves by resting cell suspensions with [³⁵S]sulfate and [¹⁴C]acetate. Both sulfate and acetate consumption followed Michaelis-Menten saturation kinetics. The half-saturation constant (K_m) for acetate uptake was 70 μM with cells from either long-term sulfate- or long-term acetate-limited chemostat cultures. The average K_m value for sulfate uptake by D. postgatei was about 200 μM. K_m values for sulfate uptake did not differ significantly when determined with cells derived either from batch cultures or sulfate- or acetate-limited chemostat cultures. Acetate consumption was observed at acetate concentrations of ≤1 μM, whereas sulfate uptake usually ceased at 5 to 20 μM. The results show that D. postgatei is not freely permeable to sulfate ions and further indicate that sulfate uptake is an energy-requiring process.     

Potential-dependent anion transport in tonoplast vesicles from oat roots

Potential-dependent anion movement into tonoplast vesicles from oat roots (Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Δψ) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H⁺-pumping pyrophosphatase, which is K⁺ stimulated and anion insensitive. The relative rate of ΔΨ dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN⁻ (100) > NO₃⁻ (72) = Cl⁻ (70) > Br⁻ (62) > SO₄²⁻ (5) = H₂PO₄⁻ (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Δψ dissipation by Cl⁻ and NO₃⁻, but not by SCN⁻, was saturable. The K_m values for Cl⁻ and NO₃⁻ uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H⁺-coupled Cl⁻ transporter on the same vesicles, the potential-dependent Cl⁻ transport was insensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl⁻ transport in these vesicles. The potentials generated by the H⁺-translocating ATPase and H⁺-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl⁻ conductance on the anion-sensitive H⁺-ATPase was found.     

Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (K_s, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (K_d, ≤3 nM) and prothioconazole-desthio the lowest (K_d, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC₅₀s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC₁₀₀, >256 μg ml⁻¹). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC₁₀₀ [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml⁻¹) with the exception of clotrimazole, which was as potent as malachite green (MIC₁₀₀, ∼1 μg ml⁻¹). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC₅₀, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC₁₀₀, ∼1 to 2 μg ml⁻¹) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.     

Properties and Expression of Na+/K+-ATPase α-Subunit Isoforms in the Brain of the Swamp Eel,Monopterus albus,Which Has Unusually High Brain Ammonia Tolerance

The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l⁻¹) and accumulate ammonia to high concentrations in its brain (∼4.5 µmol g⁻¹). Na⁺/K⁺-ATPase (Nka) is an essential transporter in brain cells, and since NH₄ ⁺ can substitute for K⁺ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K⁺ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH₄ ⁺ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na⁺/K⁺-ATPase and Na⁺/NH₄ ⁺-ATPase activities over a range of K⁺/NH₄ ⁺ concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l⁻¹ NH₄Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na⁺/NH₄ ⁺-ATPase activities were significantly lower than the Na⁺/K⁺-ATPase activities assayed at various NH₄ ⁺/K⁺ concentrations. Furthermore, the effectiveness of NH₄ ⁺ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K⁺ specificity of K⁺ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus.     

Selectivity of the Ca2+-activated and Light-dependent K+ Channels for Monovalent Cations

The ionic selectivity of the Ca²⁺-activated K⁺ channel of Aplysia neurons and of the light-dependent K⁺ channel of Pecten photoreceptors to metal and organic cations was studied. The selectivity sequence determined from reversal potential measurements is T1⁺ K⁺ > Rb⁺ > NH⁺₄ > Cs⁺ > Na⁺, Li⁺ and is identical to the sequence determined previously for voltage-dependent K⁺ channels in a variety of tissues. Our results suggest that some physical aspect of the K⁺ channel is conserved in phyllogenetically different tissues and cells.     

Metabolism of Acetate, Methanol, and Methylated Amines in Intertidal Sediments of Lowes Cove, Maine

The fates and the rates of metabolism of acetate, trimethylamine, methylamine, and methanol were examined to determine the significance of these compounds as in situ methane precursors in surface sediments of an intertidal zone in Maine. Concentrations of these potential methane precursors were generally <3 μM, with the exception of sediments containing fragments of the seaweed Ascophyllum nodosum, in which acetate was 96 μM. [2-¹⁴C]acetate turnover in all samples was rapid (turnover time <2 h), with ¹⁴CO₂ as the primary product. [¹⁴C]trimethylamine and methylamine turnover times were slower (>8 h) and were characterized by formation of both ¹⁴CH₄ and ¹⁴CO₂. Ratios of ¹⁴CH₄/¹⁴CO₂ from [¹⁴C]trimethylamine and methylamine in uninhibited sediments indicated that a significant fraction of these substrates were catabolized via a non-methanogenic process. Data from inhibition experiments involving sodium molybdate and 2-bromoethanesulfonic acid supported this interpretation. [¹⁴C]methanol was oxidized relatively slowly compared with the other substrates and was catabolized mainly to ¹⁴CO₂. Results from experiments with molybdate and 2-bromoethanesulfonic acid suggested that methanol was oxidized primarily through sulfate reduction. In Lowes Cove sediments, trimethylamine accounted for 35.1 to 61.1% of total methane production.     

Ionic Regulation for Cytokinin-dependent Betacyanin Synthesis in Amaranthus Seedlings

Potassium ions at low concentrations stimulate cytokinin-dependent betacyanin synthesis in Amaranthus tricolor seedlings more than other alkali metal ions when tested as the chloride salts. The sequence of relative stimulation is K⁺ > Rb⁺ > (Na⁺ = Li⁺). Calcium and Mg²⁺ ions are inhibitory at concentrations > 1 millimolar when tested as chlorides. Anions also have an effect on the degree of alkali metal stimulation in the order PO₄³⁻ > NO₃⁻ > Cl⁻. The high activity of phosphate may be partly due to its chelating effect on inhibitory Ca²⁺ ions, or to effects on K⁺ uptake. A mixture of Na⁺ and K⁺ in the presence of phosphate is more effective than either cation alone. This result may be due either to effects on tyrosine transport or on the potassium uptake system. Phytochrome-dependent betacyanin synthesis shows the same stimulation by Na⁺ plus K⁺. The effect of a number of inhibitors of transport systems on betacyanin accumulation is reported. The possible role of the ionic environment of cells in their metabolic regulation is discussed, particularly in relation to cytokinin action.     

Differences in SOM Decomposition and Temperature Sensitivity among Soil Aggregate Size Classes in a Temperate Grasslands

The principle of enzyme kinetics suggests that the temperature sensitivity (Q₁₀) of soil organic matter (SOM) decomposition is inversely related to organic carbon (C) quality, i.e., the C quality-temperature (CQT) hypothesis. We tested this hypothesis by performing laboratory incubation experiments with bulk soil, macroaggregates (MA, 250–2000 μm), microaggregates (MI, 53–250 μm), and mineral fractions (MF, <53 μm) collected from an Inner Mongolian temperate grassland. The results showed that temperature and aggregate size significantly affected on SOM decomposition, with notable interactive effects (P<0.0001). For 2 weeks, the decomposition rates of bulk soil and soil aggregates increased with increasing incubation temperature in the following order: MA>MF>bulk soil >MI(P <0.05). The Q₁₀ values were highest for MA, followed (in decreasing order) by bulk soil, MF, and MI. Similarly, the activation energies (E_a) for MA, bulk soil, MF, and MI were 48.47, 33.26, 27.01, and 23.18 KJ mol⁻¹, respectively. The observed significant negative correlations between Q₁₀ and C quality index in bulk soil and soil aggregates (P<0.05) suggested that the CQT hypothesis is applicable to soil aggregates. Cumulative C emission differed significantly among aggregate size classes (P <0.0001), with the largest values occurring in MA (1101 μg g⁻¹), followed by MF (976 μg g⁻¹) and MI (879 μg g⁻¹). These findings suggest that feedback from SOM decomposition in response to changing temperature is closely associated withsoil aggregation and highlights the complex responses of ecosystem C budgets to future warming scenarios.     

C3larvin Toxin,an ADP-ribosyltransferase from Paenibacillus larvae

C3larvin toxin was identified by a bioinformatic strategy as a putative mono-ADP-ribosyltransferase and a possible virulence factor from Paenibacillus larvae, which is the causative agent of American Foulbrood in honey bees. C3larvin targets RhoA as a substrate for its transferase reaction, and kinetics for both the NAD⁺ (K_m = 34 ± 12 μm) and RhoA (K_m = 17 ± 3 μm) substrates were characterized for this enzyme from the mono-ADP-ribosyltransferase C3 toxin subgroup. C3larvin is toxic to yeast when expressed in the cytoplasm, and catalytic variants of the enzyme lost the ability to kill the yeast host, indicating that the toxin exerts its lethality through its enzyme activity. A small molecule inhibitor of C3larvin enzymatic activity was discovered called M3 (K_i = 11 ± 2 μm), and to our knowledge, is the first inhibitor of transferase activity of the C3 toxin family. C3larvin was crystallized, and its crystal structure (apoenzyme) was solved to 2.3 Å resolution. C3larvin was also shown to have a different mechanism of cell entry from other C3 toxins.     

Stimulation of plasmalemma adenosine triphosphatase from oat roots by inorganic and organic cations: concentration-dependence, selectivity, and sites

K⁺-stimulated ATPase activity of a plasmalemma-enriched fraction from excised roots of oat was triphasic in the range 5 to 80 millimolar KCl. The phases obeyed Michaelis-Menten kinetics and were separated from each other by jumps or sharp breaks at about 10 and 20 millimolar. Stimulation by alkali cations was in the order K⁺ > Rb⁺ > Na⁺ > Cs⁺ > Li⁺ or in a closely related sequence. The specificity reflected differences in V_max, not in affinity (K_m⁻¹). Stimulation by the organic cations ethanolamine and choline in the interval 11 to 80 millimolar appeared monophasic rather than biphasic. Substitution on the quaternary nitrogen of the amino alcohols decreased their effectiveness, as did extension and branching of the chain. Stimulation was maximal at about pH 7 both for K⁺ and choline.