Thermodynamic and kinetic determinants of Thermotoga maritima cold shock protein stability: a structural and dynamic analysis

The cold shock protein (CSP) from hyperthermophile Thermotoga maritima (TmCSP) is only marginally stable (DeltaG(T(opt)) = 0.3 kcal/mol) at 353 K, the optimum environmental temperature (T(opt)) for T. maritima. In comparison, homologous CSPs from E. coli (DeltaG(T(opt)) = 2.2 kcal/mol) and B. subtilis (DeltaG(T(opt)) = 1.5 kcal/mol) are at least five times more stable at 310 K, the T(opt) for the mesophiles. Yet at the room temperature, TmCSP is more stable (DeltaG(T(R)) = 4.7 kcal/mol) than its homologues (DeltaG(T(R)) = 3.0 kcal/mol for E. coli CSP and DeltaG(T(R)) = 2.1 kcal/mol for B. subtilis CSP). This unique observation suggests that kinetic, rather than thermodynamic, barriers toward unfolding might help TmCSP native structure at high temperatures. Consistently, the unfolding rate of TmCSP is considerably slower than its homologues. High temperature (600 K) complete unfolding molecular dynamics (MD) simulations of TmCSP support our hypothesis and reveal an unfolding scheme unique to TmCSP. For all the studied homologues of TmCSP, the unfolding process first starts at the C-terminal region and N-terminal region unfolds in the end. But for TmCSP, both the terminals resist unfolding for consistently longer simulation times and, in the end, unfold simultaneously. In TmCSP, the C-terminal region is better fortified and has better interactions with the N-terminal region due to the charged residues, R2, E47, E49, H61, K63, and E66, being in spatial vicinity. The electrostatic interactions among these residues are unique to TmCSP. Consistently, the room temperature MD simulations show that TmCSP is more rigid at its N- and C-termini as compared to its homologues from E. coli, B. subtilis, and B. caldolyticus.     

CHANGES IN NUTRIENT CONTENT OF PALMARIA PALMATA IN RESPONSE TO VARIABLE LIGHT AND UPWELLING IN NORTHERN SPAIN1

Light has been identified as one of the main factors affecting seaweed ecophysiology. We investigated the dependence of nutrient metabolism on sun and shade light conditions and whether episodes of upwelling of nutrient‐rich subsuperficial water could reduce the summer nutrient limitation driving physiological changes in Palmaria palmata (L.) Kuntze. We measured the major nutrient pools, photosynthetic pigments, and light curves, under sun and shade conditions during a summer period when one upwelling was recorded. The redundancy analysis (RDA) produced two clear groups: sun‐ and shade‐acclimated algae. Light was the major predictive factor. Sun‐acclimated algae exhibited higher carbon (C) and lower nitrogen (N) and phosphorus (P) content in association with the storage of floridoside (main C reserve) to benefit from higher irradiance (under nutrient limitation). Among N pools, N reserves (phycoerythrin, nitrate) were a lower proportion of the total N in sun‐acclimated algae, suggesting their degradation to fulfill the N demands of the cell. The orthophosphate content was also lower in sun‐acclimated algae, indicating its utilization as a nutrient reserve. In contrast, N within cell walls and membranes and chl a contributed to a similar proportion of the total N in sun‐ and shade‐acclimated algae, suggesting a response to sustain cell integrity. Transient high nutrient concentration due to the upwelling was unrelated to the nutrient content of the thallus. The storage of C as floridoside from high light exposure was shown to be the driving force for the metabolic adjustment of P. palmata at the end of summer before the onset of dormancy.     

Mortality rates in subjects with fetal alcohol spectrum disorders and their siblings

BACKGROUND: Our objective was to estimate the mortality rate in subjects with fetal alcohol spectrum disorders (FASD) and their siblings whose FASD status was unknown. METHODS: We used the state FASD Registry to link subjects with FASD to a North Dakota birth certificate. We were able to link 304 of 486 cases (63%). We used the birth certificates to identify the mother and children born to the mother (siblings). We then searched for death certificates for both the FASD cases and their siblings. We then calculated the annual and age‐adjusted mortality rates for the siblings of the Registry cases and compared them with mortality rates from North Dakota. RESULTS: The FASD case mortality rate was 2.4%, with a 4.5% mortality rate for their sibings, accounting for 14% of all deaths when compared to the North Dakota residents matched by age and year of death. The sibling deaths accounted for 21.5% of all cause mortality matched by age and year of death. The age‐standardized mortality ratios were 4.9 for the FASD cases and 2.6 for their siblings whose FASD status was unknown. CONCLUSIONS: Mortality rates for FASD cases and their siblings were increased and represent a substantial proportion of all cause mortality in North Dakota. Prevention of FASD may be a useful strategy to decrease mortality. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Physicochemical Properties and Dissolution Studies of Dexamethasone Acetate-β-Cyclodextrin Inclusion Complexes Produced by Different Methods

Inclusion complexes between dexamethasone acetate (DMA), a poorly water soluble drug, and β-cyclodextrin (βCD) were obtained to improve the solubility and dissolution rate of this drug. Phase-solubility profile indicated that the solubility of DMA was significantly increased in the presence of βCD (33-fold) and was classified as A_L-type, indicating the 1:1 stoichiometric inclusion complexes. Solid complexes prepared by different methods (kneading, coevaporation, freeze drying) and physical mixture were characterized by differential scanning calorimetry, thermogravimetry, infrared absorption and optical microscopy. Preparation methods influenced the physicochemical properties of the products. The dissolution profiles of solid complexes were determined and compared with those DMA alone and their physical mixture, in three different mediums: simulated gastric fluid (pH 1.2), simulated intestinal fluid (pH 7.4) and distilled water. The dissolution studies showed that in all mediums DMA presented an incomplete dissolution even in four hours. In contrast, the complexes formed presented a higher dissolution rate in simulated gastric fluid (SGF pH 1.2), which indicate that these have different ionization characteristics. According to the results, the freeze–dried and kneaded products exhibited higher dissolution rates than the drug alone, in all the mediums.     

Simulated nitrogen dynamics and nitrate leaching in a perennial grass ley

A soil nitrogen model was used for a 4-year simulation of nitrogen dynamics and nitrate leaching, both during grass ley growth and after ploughing a grass ley. Model results were compared with field measurements of soil mineral-N status and leaching. A soil water and heat model provided daily values for abiotic conditions, which were used as driving variables in the nitrogen simulation. Simulated values for mineral-N levels in the soil agreed well with field data for the first 3 years of the simulation. During the final year the model predicted considerably higher levels of soil mineral-N content compared with measurements. To reach the mineral-N level measured at the time of ploughing the ley, the simulated N-uptake by plants had to be increased by 8 g N m⁻². Simulations of nitrate leaching suggested that estimates of leaching based on measurements in tile-drained plots can be considerably underestimated. Accurate quantification of leaching in tile-drained plots often requires additional information on water-flow paths. A substantial increase in simulated and measured values for the mineral-N content of the soil occurred after ploughing the ley. In the simulation, most of the increase was due to a high crop residue input and the absence of a growing crop after ploughing. Litter accumulations in the soil during the 4-year period contributed little to the increase in soil mineral-N.     

Acetylene reduction and indoleacetic acid production by Azospirillum isolates from Cactaceous plants

Azospirillum isolates were obtained from rhizosphere soil and roots of three cactaceae species growing under arid conditions. All Azospirillum isolates from rhizosphere and roots ofStenocereus pruinosus andStenocereus stellatus were identified asA. brasilense; isolates of surface-sterilized roots fromOpuntia ficus-indica were bothA. brasilense andA. lipoferum. Azospirilla per g of fresh root in the three species ranged from 70×10³ to 11×10³. The most active strains in terms of C₂H₂ reduction (25–49.6 nmol/h·ml) and indoleacetic acid (IAA) production (36.5–77 μg/ml) were those identified asA. brasilense and isolated from Stenocereus roots.A. lipoferum isolated from Opuntia roots produced low amounts of IAA (6.5–17.5 μg/ml) and low C₂H₂-reduction activity (17.8–21.2 nmol/h·ml).     

Effects of CaSO4 and NaCl on growth and nitrogen fixation ofLeucaena leucocephala

Effects and interactions of varying CaSO₄ and NaCl levels on growth and nitrogen fixation ofLeucaena leucocephala K8 were examined. Leucaena was grown in nutrient solution at four levels of CaSO₄ (0.5, 1.0, 2.5 and 5.0 mM) and NaCl (1, 25, 50 and 100 mM) in randomized blocks with five replications. While NaCl significantly reduced plant growth, additions of CaSO₄ increased plant height, leaf number, and biomass of salt treated plants. For the nonsaline treatments, high CaSO₄ levels slightly depressed growth, which contradicts suggestions that Leucaena has a high calcium requirement. A significant calcium/sodium interaction was not seen for nodule number or weight. Nodule number was significantly depressed by 100 mM NaCl and nodule weight of the salt stressed plants significantly increased as CaSO₄ concentration increased from 0.5 to 2.5 mM. Effects of NaCl and CaSO₄ on nitrogen content of plant parts were inconclusive. The promotion of Leucaena salinity tolerance by addition of CaSO₄ may be attributed to the effect of calcium in maintaing the selective permeability of membranes.     

A rapid non-destructive assay to quantify soybean nodule gas permeability

The low gas permeability of a diffusion barrier in the cortex of soybean nodules plays a significant role in the protection of nitrogenase from oxygen inactivation. It may also set an upper limit on nodule respiration and nitrogen fixation rates. Two methods which have been used to quantify the gas permeability of leguminous nodules are reviewed and found to be unreliable. A new assay technique for determining both the nodule activity and gas permeability is developed and tested. This ‘lag-phase’ assay is based on the time nodules require to reach steady-state ethylene production after being exposed to acetylene. The technique is rapid, insensitive to errors in biochemical parameters associated with nitrogenase, and is non-destructive. The method was tested with intact aeroponically grown soybean plants for which the mean nodule gas permeability was found to be 13.3×10⁻³ mms⁻¹. This corresponds to a layer of cells approximately 35 um thick and is consistent with previously reported values.     

Molecular cloning, characterization and regulation of two different NADH-glutamate synthase cDNAs in bean nodules

NADH-dependent glutamate synthase (NADH-GOGAT) is a key enzyme in primary ammonia assimilation in Phaseolus vulgaris nodules. Two different types of cDNA clones of PvNADH-GOGAT were isolated from the nodule cDNA libraries. The full-length cDNA clones of PvNADH-GOGAT-I (7.4 kb) and PvNADH-GOGAT-II (7.0 kb), which displayed an 83% homology between them, were isolated using cDNA library screening, 'cDNA library walking' and RT-PCR amplification. Southern analysis employing specific 5' cDNA probes derived from PvNADH-GOGAT-I and PvNADH-GOGAT-II indicated the existence of a single copy of each gene in the bean genome. Both these proteins contain ∼100 amino acid sequences theoretically addressing each isoenzyme to different subcellular compartments. RT-PCR analysis indicated that PvNADH-GOGAT-II expression is higher than PvNADH-GOGAT-I during nodule development. Expression analysis by RT-PCR also revealed that both of these genes are differentially regulated by sucrose. On the other hand, the expression of PvNADH-GOGAT-I , but not PvNADH-GOGAT-II, was inhibited with nitrogen compounds. In situ hybridization and promoter expression analyses demonstrated that the NADH-GOGAT-I and -II genes are differentially expressed in bean root and nodule tissues. In silico analyses of the NADH-GOGAT promoters revealed the presence of potential cis elements in them that could mediate differential tissue-specific, and sugar and amino acid responsive expression of these genes.     

Dry nitrogen deposition estimates over a forest experiencing free air CO2 enrichment

The quantification of atmospheric additions of nitrogen (N) to an ecosystem is often desirable, but difficult for many locations including many ecological manipulation experiments. Ideal methodologies for the complete and chemically speciated quantification of dry N deposition (e.g. tunable diode laser absorption spectrometer, thermal‐dissociation laser‐induced fluorescence) are expensive, technologically challenging to maintain, and rarely colocated with important global change manipulation experiments. Here we present an alternative method for obtaining an approximation of total N deposition using short‐term eddy flux and concentration measurements, annual regional concentration estimates, and modeling for the Duke Experimental Forest. The motivation for generating estimates for this location was to inform the long‐term elevated CO₂ experiment conducted at Duke Forest. We estimated the total annual atmospheric N deposition to the forest to be 13.7 kg N ha^?1. Of this total, ～58% was in dry‐deposited forms. Surprisingly, and contrary to some previous predictions, nitric acid (HNO₃) was not the dominant portion of the total dry‐deposited N, implying strongly that other forms of gaseous N like organic peroxy and alkyl nitrate compounds are a significant portion of the total flux. Furthermore, this study sheds light on the completeness of the estimates derived from Clean Air Status and Trends Network (CASTNet) and other dry deposition networks. CASTNet does not measure organic forms of dry deposition. In fact, CASTNet only quantifies HNO₃ in the gas phase and may significantly underestimate total N deposition in many environments.