Osmoprotectant β-alanine betaine synthesis in the Plumbaginaceae: S-adenosyl- l-methionine dependent N-methylation of β-alanine to its betaine is via N-methyl and N,N-dimethyl β-alanines

β -Alanine betaine is an osmoprotective compound accumulated by most members of the plant family Plumbaginaceae. Leaf and root tissues of Limonium latifolium known to accumulate β -alanine betaine readily convert supplied β -alanine to β -alanine betaine. To identify the intermediates and the enzymes involved in β -alanine betaine synthesis, radiotracer experiments using [ 14 C] formate were employed. These studies demonstrate that β -alanine betaine is synthesized from β -alanine via N -methyl and N,N- dimethyl β -alanines. A rapid and sensitive radiometric assay was developed to measure N -methyltransferase (NMT) activities by using [methyl-¹⁴C] or [methyl-³H] S -adenosyl- l -methionine (AdoMet) as the methyl donor. Leaf extracts from β -alanine betaine accumulators – Armeria maritima , L. latifolium and L. ramosissimum – had detectable NMT activities while none were found in L. perezii , a species that does not accumulate β -alanine betaine. The NMT activities were further characterized from the leaves of L. latifolium . The activities had a pH optimum of 8.0, were soluble and inhibited by S -adenosyl- l -homocysteine. Extractable activities were similar from plants grown under control and salinity stress conditions. Radiolabeling with [ 14 C] l -aspartic acid indicated that, unlike in bacteria, decarboxylation of l -aspartic acid is not the source of β -alanine in the Plumbaginaceae.     

Beef cattle pasture to wetland reconversion: Impact on soil organic carbon and phosphorus dynamics

There is a major need to understand the historical condition and chemical/biological functions of the ecosystems following a conversion of wetlands to agricultural functions. To better understand the dynamics of soil total organic carbon (TOC) and phosphorus (P) during beef cattle pastures to wetland reconversion, soil core samples were collected from the beef cattle pasture and from the natural wetland at Plant City, FL, during five summer seasons (2002–2007). The levels of TOC and soil P were significantly affected by changing land use and hydrology. Draining natural wetlands to grazed pastures resulted in very pronounced reduction of TOC from 180.1 to 5.4 g g^?1. Cumulative concentrations of total phosphorus (TP) in soils (1134 mg kg^?1) under drained condition are two to three times lower than those in soils (2752 mg kg^?1) under flooded condition over the periods of land use reconversion. There was a declining trend (r = 0.82**; p ≤ 0.01) in total soil P from natural wetland (763 mg kg^?1) to altered pastures (340 mg kg^?1), largely as organic-bound P (natural wetland, 48%; grazed pastures, 44%; altered pastures, 29%). These results are important in establishing baseline information on soil properties in pasture and wetland prior to restoring and reconverting pasture back to wetland conditions. The results further suggest that changes in soil properties due to changing land use and hydrologic conditions (drying and re-wetting) could be long lasting.     

Binding and the nature of Cu(II) ion interaction with nucleosomes

The energetics of Cu (II) ion binding to mononucleosomes from C3HA mice liver and ascitic hepatoma 22A cells was determined from their binding isotherms by equilibrium dialysis and pulse high frequency inductively coupled plasma atomic emission spectroscopy. Anticooperative binding of copper ions with normal and tumor mononucleosomes were observed under various NaCl concentrations (0.002; 0.02; 0.2 M). The binding constants of Cu(II) ion with normal mononucleosomes in 0.002, 0.02, 0.2 M NaCl are 6.10×10⁴,5.22×10⁴,4.31×10⁴ respectively. The binding constants of Cu(II) ion with tumor mononucleosomes in 0.002, 0.02, 0.2 M NaCl are 6.68×10⁴,6.12×10⁴,4.82×10⁴ respectively.     

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5''-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 °C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.     

Effect of cigarette smoke exposure and structural modifications on the α-1 Antitrypsin interaction with caspases

α-1 Antitrypsin (A1AT) is a serpin with a major protective effect against cigarette smoke-induced emphysema development, and patients with mutations of the A1AT gene display a markedly increased risk for developing emphysema. We reported that A1AT protects lung endothelial cells from apoptosis and inhibits caspase-3 activity. It is not clear if cigarette smoking or A1AT mutations alter the caspase-3 inhibitory activity of A1AT and if this serpin alters the function of other caspases. We tested the hypothesis that the caspase-3 inhibitory activity of A1AT is impaired by cigarette smoking and that the A1AT RCL, the key antiprotease domain of the serpin, is required for its interaction with the caspase. We examined the caspase-3 inhibitory activity of human A1AT purified from plasma of actively smoking and nonsmoking individuals, either affected or unaffected with chronic obstructive pulmonary disease. We also tested the caspase inhibitory activity of two mutant forms of A1AT, the recombinant human piZZ and the RCL-deleted (RCL-null) A1AT forms. A1AT purified from the blood of active smokers exhibited marked attenuation in its caspase-3 inhibitory activity, independent of disease status. In vitro exposure of the normal (MM) form of A1AT to cigarette smoke extract reduced its ability to interact with caspase-3, measured by isothermal titration calorimetry, as did the deletion of the RCL, but not the ZZ point mutation. In cell-free assays A1AT was capable of inhibiting all executioner caspases, -3, -7 and especially -6, but not the initiator or inflammatory caspases. The inhibitory effect of A1AT against caspase-6 was tested in vivo, where overexpression of both human MM and ZZ-A1AT via adeno-associated virus transduction significantly protected against apoptosis and against airspace damage induced by intratracheal instillation of caspase-6 in mice. These data indicate a specific inhibitory effect of A1AT on executioner caspases, which is profoundly attenuated by active exposure to cigarette smoking and is dependent on the protein RCL, but is not affected by the PiZZ mutation.     

beta-Alanine betaine synthesis in the Plumbaginaceae. Purification and characterization of a trifunctional, S-adenosyl-L-methionine-dependent N-methyltransferase from Limonium latifolium leaves.

beta-Alanine (beta-Ala) betaine is an osmoprotective compound accumulated by most members of the highly stress-tolerant family Plumbaginaceae. Its potential role in plant tolerance to salinity and hypoxia makes its synthetic pathway an interesting target for metabolic engineering. In the Plumbaginaceae, beta-Ala betaine is synthesized by S-adenosyl-L-methionine-dependent N-methylation of beta-Ala via N-methyl beta-Ala and N,N-dimethyl beta-Ala. It was not known how many N-methyltransferases (NMTases) participate in the three N-methylations of beta-Ala. An NMTase was purified about 1,890-fold, from Limonium latifolium leaves, using a protocol consisting of polyethylene glycol precipitation, heat treatment, anion-exchange chromatography, gel filtration, native polyacrylamide gel electrophoresis, and two substrate affinity chromatography steps. The purified NMTase was trifunctional, methylating beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that the native NMTase is a dimer of 43-kD subunits. The NMTase had an apparent K(m) of 45 microM S-adenosyl-l-methionine and substrate inhibition was observed above 200 microM. The apparent K(m) values for the methyl acceptor substrates were 5.3, 5.7, and 5.9 mM for beta-Ala, N-methyl beta-Ala, and N,N-dimethyl beta-Ala, respectively. The NMTase had an isoelectric point of 5.15 and was reversibly inhibited by the thiol reagent p-hydroxymercuribenzoic acid.     

Genetic Diversity and Geographical Distribution of Indigenous Soybean-Nodulating Bradyrhizobia in the United States

We investigated the relationship between the genetic diversity of indigenous soybean-nodulating bradyrhizobia and their geographical distribution in the United States using nine soil isolates from eight states. The bradyrhizobia were inoculated on three soybean Rj genotypes (non-Rj, Rj₂Rj₃, and Rj₄). We analyzed their genetic diversity and community structure by means of restriction fragment length polymorphisms of PCR amplicons to target the 16S-23S rRNA gene internal transcribed spacer region, using 11 USDA Bradyrhizobium strains as reference strains. We also performed diversity analysis, multidimensional scaling analysis based on the Bray-Curtis index, and polar ordination analysis to describe the structure and geographical distribution of the soybean-nodulating bradyrhizobial community. The major clusters were Bradyrhizobium japonicum Bj123, in the northern United States, and Bradyrhizobium elkanii, in the middle to southern regions. Dominance of bradyrhizobia in a community was generally larger for the cluster belonging to B. elkanii than for the cluster belonging to B. japonicum. The indigenous American soybean-nodulating bradyrhizobial community structure was strongly correlated with latitude. Our results suggest that this community varies geographically.