Continuous-time modeling of cell fate determination in Arabidopsis flowers

Background  

The genetic control of floral organ specification is currently being investigated by various approaches, both experimentally and through modeling. Models and simulations have mostly involved boolean or related methods, and so far a quantitative, continuous-time approach has not been explored.     

Methanogenium marinum sp. nov., a H2-using methanogen from Skan Bay,Alaska, and kinetics of H2 utilization

A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska. The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 μm), occurring singly. Cells grew by reducing CO₂ with H₂ or formate as electron donor. Growth on formate was much slower than that on H₂. Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized. The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B₁₂, and peptones for growth; yeast extract stimulated growth but was not required. The cells grew fastest at 25 °C (range 5 °C to 25 °C), at a pH of 6.0 – 6.6 (growth range, pH 5.5 – 7.5), and at a salinity of 0.25 – 1.25 M Na⁺. Cells of this and other H₂-using methanogens from saline environments metabolized H₂ to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure. We propose that the threshold pressure may be limited by the energetics of catabolism. The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogenium frigidum Ace-2. DNA–DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2. These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium. Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this. Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain. This revised version was published online in August 2006 with corrections to the Cover Date.     

Potassium modulation of methionine uptake in astrocytes in vitro

Methionine participates in a large variety of metabolic pathways in brain, and its transport may play an important regulatory role. The properties of methionine uptake were examined in a preparation of neonatal rat brain astrocytes. Uptake is linear for 15 minutes, up to 2.5 M. At steady state conditions, methionine is concentrated 30–50-fold. Measured methionine homoexchange accounts for a significant fraction of uptake at concentrations greater than 10 M. We recently reported that methionine uptake is decreased by elevations in extracellular K⁺. Potassium induced efflux cannot account for this apparent effect; and thus for concentrations less than 2.5M, and for short times of incubation, measured rates of methionine uptake represent unidirectional flux. At extracellular concentrations of K⁺ equal to 6.9 mM, the apparentV _max of methionine transport is 182 pmol/min/mg protein, and theK _m is 1.3 M. Where K⁺ is shifted to 11.9 mM, theK _m remains unchanged, and theV _max is reduced by half.     

The effect of root temperature on the induction of nitrate reductase activities and nitrogen uptake rates in arctic plant species

We investigated whether six arctic plant species have the potential to induce nitrate reductase (NR) activity when exposed to NO₃ ^--nitrogen under controlled environment conditions, using an in vivo assay that uses the rate of NO₂ ^--accumulation to estimate potential NR activity. We also assessed the effect of low root temperatures on NR activity, growth and nitrogen uptake (using ¹⁵N applications) in two of the selected species. Five of the six species (Cerastium alpinum, Dryas intergrifolia, Oxyria digyna, Saxifraga cernua and Salix arctica) were capable of inducing NR activity when exposed to solutions containing 0.5 mM NO₃ ^- at 20°C for 10 days. Although in vivo NR activity was not induced in Saxifraga oppositifolia under controlled conditions, we conclude that it was capable of growing successfully on NO₃ ^-, due to the presence of moderate rates of NR activity observed in both NH₄ ⁺-grown and NO₃ ^--treated plants. Exposure of O. digyna and D. integrifolia to 3°C root temperatures for two weeks, with the shoots kept at 20°C, resulted in root and leaf NR activity rates of NO₃ ^--treated plants being reduced to rates exhibited by NH₄ ⁺-grown plants. Although these decreases in NR in both species appeared to be due to limitations in NO₃ ^--uptake and growth rate (rather than direct low-temperature inhibition of NR synthesis per se), direct low-temperature inhibition of root NR synthesis could not be ruled out. In contrast to the temperature insensitivity of NH₄ ⁺ uptake in D. integrifolia, NO₃ ^--uptake in D. integrifolia was inhibited by low root temperatures. We conclude that the selected arctic species have the genetic potential to utilize NO₃ ^--nitrogen, and that low root temperatures, in conjunction with other environmental limitations, may be responsible for the lack of induction of NR in D. integrifolia and Salix arctica under field conditions.     

Cloning,characterization and regulation of a family of phi class glutathione transferases from wheat

Six phi (F) class glutathione transferases (GSTs) were cloned from bread wheat (Triticum aestivum L.) treated with the herbicide safener fenchlorazole ethyl and named TaGSTF1–6. Recombinant TaGSTFs were assayed for glutathione conjugating activity towards xenobiotics including herbicides and for glutathione peroxidase (GPOX) activity. TaGSTF1, which resembled ZmGSTF1, the dominant GST in maize (Zea mays), was highly active in conjugating 1-chloro-2,4-dinitrobenezene (CDNB) but had low activities towards chloroacetanilide, diphenyl ether and aryloxphenoxypropionate herbicides. TaGSTF2, TaGSTF3 and TaGSTF4 all resembled the safener-inducible ZmGSTF2, with TaGSTF2 and TaGSTF3 being highly active GPOXs and rapidly detoxifying chloroacetanilides. TaGSTF5 resembled ZmGSTF3, having limited conjugating and GPOX activity. TaGSTF6 contained both ZmGSTF1- and ZmGSTF2-like sequences but was most similar to ZmGSTF1 in detoxifying activity. The expression of TaGSTFs in wheat seedlings was enhanced upon exposure to fenchlorazole ethyl, herbicides or other chemical inducing treatments. TaGSTFs were also enhanced by treatment with the natural products caffeic acid, 7,4-dihydroxyflavone and naringenin. The CDNB-conjugating activity of TaGSTF1, and to a lesser extent TaGSTF6, was highly sensitive to inhibition by flavonoids, particularly the chalcone isoliquiritigenin. The other TaGSTFs were much less sensitive to such inhibition. It was subsequently determined that isoliquiritigenin underwent glutathione conjugation, though this reversible reaction did not require the intervention of any TaGSTF. The potential importance of GSTFs and glutathione conjugation in flavonoid metabolism is discussed.     

Determining the origin of the stabilization of DNA by 5-aminopropynylation of pyrimidines

DNA duplexes are stabilized by aminopropynyl modification of pyrimidines at the 5 position. A combination of thermodynamic analyses as a function of ionic strength, NMR, and molecular modeling has been applied to determine the origin of the stabilization. UV melting studies of a dodecamer bearing one, two, or three nonadjacent modified dU and dC and of a single dU(8) in the Dickerson-Drew dodecamer revealed that the modifications are essentially additive in terms of T(m), DeltaG, and DeltaH, and there is little difference between dU and dC. The free energy change was parsed into electrostatic and nonelectrostatic components, which showed a significant contribution from charge interactions at physiological ionic strength but also a nonelectrostatic contribution that arises in part from hydration. NMR spectroscopy of the modified Dickerson-Drew dodecamer revealed that the conformation of the duplexes is not significantly altered by the modifications, though (31)P NMR shows that the positive charge may affect ionic interactions with the oxygen atoms of the neighboring phosphates. The modified duplex showed significant hydration in both major and minor grooves. The single strands were also analyzed by NMR, which showed evidence of significant stacking interactions in the modified oligonucleotide. Parsing the energy contribution has shown that electrostatics and hydration can produce substantial increases in thermodynamic stability without significant changes in the conformation of the duplex state. These considerations have significance for the design of oligonucleotides used for hybridization.     

Synthesis and study of the fluorescein conjugate of the nucleotide dPTP

The synthesis of a fluorescein conjugate on the non-natural base P is described. The ability of the newly synthesised fluorescein--dPTP and of a fluorescein-11-dUTP to compete with the natural nucleotide TTP was also studied. Overall the efficiency of labelling a nucleic acid with a fluorescein moiety was found to be approximately equal.