Leiomodin creates a leaky cap at the pointed end of actin-thin filaments.

Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a "swinging gate" allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance.

Studies of the Complexation of Gluconate with Th(IV) in Acidic Solutions: Stability Constant Determination and Coordination Mode Analysis.

Gluconate is a multidentate ligand and its complexation with actinides has received increasing attention because of its existence in high level nuclear wastes as well as in nuclear waste repositories. In this work, the complexation of gluconate with Th(IV) was studied in deuterated water (D2O) by pD titrations and nuclear magnetic resonance (NMR) spectroscopy. In the pCD range 2.0-4.6, gluconate (GD4-) forms two 1:1 complexes with Th(IV), Th(GD3)2+ and Th(GD2)+. Their stability constants were determined to be log ß(D)101(-1) = 1.04 ± 0.12 for Th4+ + GD4- = Th(GD3)3+ + D+ and log ß(D)101(-2) = -(1.31 ± 0.09) for Th4+ + GD4- = Th(GD2)+ + 2D+ at I = 1.0 mol·L-1 NaClO4 and t = 22 °C. The coordination modes of these two complexes were also analyzed. In both complexes, the tridentate chelation forms through the binding of Th(IV) to one oxygen from the carboxylate group and two oxygens from α- and γ-hydroxyl groups. The difference is that in Th(GD2)+, both α- and γ-hydroxyl groups deprotonate, and in Th(GD3)2+, only the α-hydroxyl group deprotonates.

Localization of the binding interface between leiomodin-2 and α-tropomyosin.

The development of some familial dilated cardiomyopathies (DCM) correlates with the presence of mutations in proteins that regulate the organization and function of thin filaments in cardiac muscle cells. Harmful effects of some mutations might be caused by disruption of yet uncharacterized protein-protein interactions. We used nuclear magnetic resonance spectroscopy to localize the region of striated muscle α-tropomyosin (Tpm1.1) that interacts with leiomodin-2 (Lmod2), a member of tropomodulin (Tmod) family of actin-binding proteins. We found that 21 N-terminal residues of Tpm1.1 are involved in interactions with residues 7-41 of Lmod2. The K15N mutation in Tpm1.1, known to be associated with familial DCM, is located within the newly identified Lmod2 binding site of Tpm1.1. We studied the effect of this mutation on binding Lmod2 and Tmod1. The mutation reduced binding affinity for both Lmod2 and Tmod1, which are responsible for correct lengths of thin filaments. The effect of the K15N mutation on Tpm1.1 binding to Lmod2 and Tmod1 provides a molecular rationale for the development of familial DCM.

Keeping PASE with WEFT: SHWEFT-PASE pulse sequences for 1H NMR spectra of highly paramagnetic molecules.

Metalloproteins are a category of biomolecules in which the metal site is usually the locus of activity or function. In many cases, the metal ions are paramagnetic or have accessible paramagnetic states, many of which can be studied using NMR spectroscopy. Extracting useful information from (1)H NMR spectra of highly paramagnetic proteins can be difficult because the paramagnetism leads to large resonance shifts (~400 ppm), extremely broad lines, extreme baseline nonlinearity, and peak shape distortion. It is demonstrated that employing polychromatic and adiabatic shaped pulses in simple pulse sequences, then combining existing sequences, leads to significant spectral improvement for highly paramagnetic proteins. These sequences employ existing technology, with available hardware, and are of short duration to accommodate short nuclear T1 and T2. They are shown to display uniform excitation over large spectral widths (~75 kHz), accommodate high repetition rates, produce flat baselines over 75 kHz while maintaining peak shape fidelity, and can be used to reduce spectral dynamic range. High-spin (S = 5/2) metmyoglobin, a prototypical highly paramagnetic protein, was used as the test molecule. The resulting one-dimensional (1D) pulse sequences combine shaped pulses with super-water elimination Fourier transform, which can be further combined with paramagnetic spectroscopy to give shaped pulses with super-water elimination Fourier transform-paramagnetic spectroscopy. These sequences require, at most, direct current offset correction and minimal phasing. The performance of these sequences in simple (1)H 1D, 1D NOE, and two-dimensional NOESY experiments is demonstrated for metmyoglobin and Paracoccus denitrificans Co(2+)-amicyanin (S = 3/2), and employed to make new heme hyperfine resonance assignments for high-spin metBjFixLH(151-256), the heme sensing domain of Bradyrhizobium japonicum FixL.

In vitro phosphinate methylation by PhpK from Kitasatospora phosalacinea.

Radical S-adenosyl-L-methionine, cobalamin-dependent methyltransferases have been proposed to catalyze the methylations of unreactive carbon or phosphorus atoms in antibiotic biosynthetic pathways. To date, none of these enzymes has been purified or shown to be active in vitro. Here we demonstrate the activity of the P-methyltransferase enzyme, PhpK, from the phosalacine producer Kitasatospora phosalacinea. PhpK catalyzes the transfer of a methyl group from methylcobalamin to 2-acetylamino-4-hydroxyphosphinylbutanoate (N-acetyldemethylphosphinothricin) to form 2-acetylamino-4-hydroxymethylphosphinylbutanoate (N-acetylphosphinothricin). This transformation gives rise to the only carbon-phosphorus-carbon linkage known to occur in nature.

Sequence-controlled oligomers fold into nanosolenoids and impart unusual optical properties.

Controlled syntheses give unique block oligomers with alternating flexible ethylene glycol and rigid perylenetetracarboxylic diimide (PDI) units. The number of rigid units vary from n=1 to 10. PDI units were stitched together by using efficient phosphoramidite chemistry. The resulting oligomers undergo folding in most solvents, including chloroform. In their ground state, these folded oligomers were characterized by using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), NMR spectroscopy, and electronic absorption spectroscopy. FTICR-MS revealed the exact masses of these sequence-controlled oligomers, which confirmed the chemical composition and validated the synthetic strategy. The NMR neighboring ring-current effect (NRE) indicates the formation of cofacial π stacks; the stacked aromatic rings have nearly coaxial alignment akin to a nanosoleniod. Nanosolenoidal shielding in π stacks causes all aromatic protons to shift upfield, whereas NOE in a cyclic hetero-chromophoric dimer supports a rotated, cofacial π-stacking orientation separated by about 3.5 Å. Electron-phonon coupling is much stronger than excitonic coupling in these self-folded PDI oligomers; thus, Franck-Condon factors dictate the observed spectral features in visible spectra. The absorbance spectrum exhibits weak hypochromism due to π stacking with increasing stacking units n. Finally, ab initio calculations support the experimental observations, indicating 3.5 Å cofacial spacing in which one molecule is rotated 30° from the eclipsed orientation and higher oligomers can adopt, without a compensating energy penalty, either the right/left-handed helices or the 1,3-eclipsed structures. Both theory and experiments validate the nano-π-solenoids and their novel photophysical properties.

Methodology for scenario-based assessments and demonstration of treatment effectiveness using the Leaching Environmental Assessment Framework (LEAF).

A methodology for developing scenario-based leaching assessments as part of the Leaching Environmental Assessment Framework (LEAF) is illustrated using a hypothetical management/treatment scenario of contaminated soil from a copper and lead smelter. Scenario assessments refine the process beyond screening-level assessments by considering site- and scenario-specific information about the disposal or utilization environment. LEAF assessments assume (i) granular materials leach at local equilibrium with percolating water, while (ii) monolithic materials (e.g., low permeability solidified/stabilized soils) leach by diffusion-based mass transport toward surrounding contact water. Leaching concentrations, estimated using LEAF leaching test data and estimated or measured scenario information, are compared to threshold values. Demonstration results indicate that leaching from untreated soil is significantly (>10x) greater from solidified/stabilized soil than treated material, except for highly soluble constituents (Cl-, NO3-2) or when constituents have similar equilibrium concentrations in both materials (As, Pb). Comparison between wet and dry environments show that while dry environments lead to lower COPC mass-based rates of leaching, the leaching concentrations may be higher due to lower liquid-to-solid ratios. The presented assessment methodology can be used to evaluate treatment effectiveness when both physical and chemical retention characteristics of the material are altered.

Demonstration of the use of test results from the Leaching Environmental Assessment Framework (LEAF) to develop screening-level leaching assessments.

Environmental management often benefits from leaching assessment as a predictive tool for estimating constituent leaching from solid and waste materials. The Leaching Environmental Assessment Framework (LEAF) provides both validated tests methods for characterizing materials and a methodology for developing screening assessments based on material characterization results. The use of LEAF data in a screening-level environmental assessment approach is demonstrated through a hypothetical case study of copper/lead smelter soil remediation. The LEAF test methods characterize leaching behavior from a wide range of materials as either constituent liquid-solid partitioning as functions of pH and liquid-to-solid ratio (L/S) or as a rate of constituent mass transport. In this study, leaching characteristics of a contaminated smelter soil and the same soil treated by solidification/stabilization with Portland cement were compared to hypothetical environmental thresholds. Screening assessments were developed for total content, available content, and maximum concentrations over relevant pH domains and L/S ranges. Assessment ratios for barium, beryllium, and fluoride indicated that estimated leaching would be less than thresholds in both materials and these constituents were removed from further analysis. Similarly, chromium (in soil) and zinc (in solidified material) were screened from further analysis. For the remaining constituents, scenario-based assessment could refine estimated leaching concentrations by considering anticipated conditions of leaching scenario.

Evaluating the fate of metals in air pollution control residues from coal-fired power plants.

Changes in emissions control at U.S. coal-fired power plants will shift metals content from the flue gas to the air pollution control (APC) residues. To determine the potential fate of metals that are captured through use of enhanced APC practices, the leaching behavior of 73 APC residues was characterized following the approach of the Leaching Environmental Assessment Framework. Materials were tested over pH conditions and liquid-solid ratios expected during management via land disposal or beneficial use. Leachate concentrations for most metals were highly variable over a range of coal rank, facility configurations, and APC residue types. Liquid-solid partitioning (equilibrium) as a function of pH showed significantly different leaching behavior for similar residue types and facility configurations. Within a facility, the leaching behavior of blended residues was shown to follow one of four characteristic patterns. Variability in metals leaching was greater than the variability in totals concentrations by several orders of magnitude, inferring that total content is not predictive of leaching behavior. The complex leaching behavior and lack of correlation to total contents indicates that release evaluation under likely field conditions is a better descriptor of environmental performance than totals content or linear partitioning approaches.

Evelynin, a cytotoxic benzoquinone-type Retro-dihydrochalcone from Tacca chantrieri.

A new benzoquinone-type retro-dihydrochalcone, named evelynin, was isolated from the roots and rhizomes of Tacca chantrieri. The structure was elucidated on the basis of the analysis of spectroscopic data and confirmed by a simple one-step total synthesis. Evelynin exhibited cytotoxicity against four human cancer cell lines, MDA-MB-435 melanoma, MDA-MB-231 breast, PC-3 prostate, and HeLa cervical carcinoma cells, with IC(50) values of 4.1, 3.9, 4.7, and 6.3 µM, respectively.

Complexation of uranium(VI) by gluconate in acidic solutions: a thermodynamic study with structural analysis.

Within the pC(H) range of 2.5 to 4.2, gluconate forms three uranyl complexes UO(2)(GH(4))(+), UO(2)(GH(3))(aq), and UO(2)(GH(3))(GH(4))(-), through the following reactions: (1) UO(2)(2+) + GH(4)(-) = UO(2)(GH(4))(+), (2) UO(2)(2+) + GH(4)(-) = UO(2)(GH(3))(aq) + H(+), and (3) UO(2)(2+) + 2GH(4)(-) = UO(2)(GH(3))(GH(4))(-) + H(+). Complexes were inferred from potentiometric, calorimetric, NMR, and EXAFS studies. Correspondingly, the stability constants and enthalpies were determined to be log beta(1) = 2.2 +/- 0.3 and DeltaH(1) = 7.5 +/- 1.3 kJ mol(-1) for reaction (1), log beta(2) = -(0.38 +/- 0.05) and DeltaH(2) = 15.4 +/- 0.3 kJ mol(-1) for reaction (2), and log beta(3) = 1.3 +/- 0.2 and DeltaH(3) = 14.6 +/- 0.3 kJ mol(-1) for reaction (3), at I = 1.0 M NaClO(4) and t = 25 degrees C. The UO(2)(GH(4))(+) complex forms through the bidentate carboxylate binding to U(VI). In the UO(2)(GH(3))(aq) complex, hydroxyl-deprotonated gluconate (GH(3)(2-)) coordinates to U(VI) through the five-membered ring chelation. For the UO(2)(GH(3))(GH(4))(-) complex, multiple coordination modes are suggested. These results are discussed in the context of trivalent and pentavalent actinide complexation by gluconate.

Evidence of cyclical light/dark-regulated expression of freezing tolerance in young winter wheat plants.

The ability of winter wheat (Triticum aestivum L.) plants to develop freezing tolerance through cold acclimation is a complex rait that responds to many environmental cues including day length and temperature. A large part of the freezing tolerance is conditioned by the C-repeat binding factor (CBF) gene regulon. We investigated whether the level of freezing tolerance of 12 winter wheat lines varied throughout the day and night in plants grown under a constant low temperature and a 12-hour photoperiod. Freezing tolerance was significantly greater (P<0.0001) when exposure to subfreezing temperatures began at the midpoint of the light period, or the midpoint of the dark period, compared to the end of either period, with an average of 21.3% improvement in survival. Thus, freezing survival was related to the photoperiod, but cycled from low, to high, to low within each 12-hour light period and within each 12-hour dark period, indicating ultradian cyclic variation of freezing tolerance. Quantitative real-time PCR analysis of expression levels of CBF genes 14 and 15 indicated that expression of these two genes also varied cyclically, but essentially 180° out of phase with each other. Proton nuclear magnetic resonance analysis (1H-NMR) showed that the chemical composition of the wheat plants' cellular fluid varied diurnally, with consistent separation of the light and dark phases of growth. A compound identified as glutamine was consistently found in greater concentration in a strongly freezing-tolerant wheat line, compared to moderately and poorly freezing-tolerant lines. The glutamine also varied in ultradian fashion in the freezing-tolerant wheat line, consistent with the ultradian variation in freezing tolerance, but did not vary in the less-tolerant lines. These results suggest at least two distinct signaling pathways, one conditioning freezing tolerance in the light, and one conditioning freezing tolerance in the dark; both are at least partially under the control of the CBF regulon.

Characterization of coffee (Coffea arabica) husk lignin and degradation products obtained after oxygen and alkali addition.

The full use of biomass in future biorefineries has stimulated studies on utilization of lignin from agricultural crops, such as coffee husk, a major residue from coffee processing. This study focuses on characterizing the lignin obtained from coffee husk and its further wet oxidation products as a function of alkali loading, temperature and residence time. The lignin fraction after diluted acid and alkali pretreatments is composed primarily of p-hydroxylphenyl units (≥49%), with fewer guaiacyl and syringyl units. Linkages appear to be mainly ß-O-4 ether linkages. Thermal degradation of pretreated lignin during wet oxidation occurred in two stages. Carboxylic acids were the main degradation product. Due to the condensed structure of this lignin, relatively low yields of aromatic aldehydes were achieved, except with temperatures over 210 °C, 5 min residence time and 11.7 wt% NaOH. Optimization of the pretreatment and oxidation parameters are important to maximizing yield of high-value bioproducts from lignin.

The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.

Oxidative degradation of biorefinery lignin obtained after pretreatment of forest residues of Douglas Fir.

Harvested forest residues are usually considered a fire hazards and used as "hog-fuel" which results in air pollution. In this study, the biorefinery lignin stream obtained after wet explosion pretreatment and enzymatic hydrolysis of forestry residues of Douglas Fir (FS-10) was characterized and further wet oxidized under alkaline conditions. The studies indicated that at 10% solids, 11.7wt% alkali and 15min residence time, maximum yields were obtained for glucose (12.9wt%), vanillin (0.4wt%) at 230°C; formic acid (11.6wt%) at 250°C; acetic acid (10.7wt%), hydroxybenzaldehyde (0.2wt%), syringaldehyde (0.13wt%) at 280°C; and lactic acid (12.4wt%) at 300°C. FTIR analysis of the solid residue after wet oxidation showed that the aromatic skeletal vibrations relating to lignin compounds increased with temperature indicating that higher severity could result in increased lignin oxidation products. The results obtained, as part of the study, is significant for understanding and optimizing processes for producing high-value bioproducts from forestry residues.

Characterization in vitro and in vivo of the putative multigene 4-coumarate:CoA ligase network in Arabidopsis: syringyl lignin and sinapate/sinapyl alcohol derivative formation.

A recent in silico analysis revealed that the Arabidopsis genome has 14 genes annotated as putative 4-coumarate:CoA ligase isoforms or homologues. Of these, 11 were selected for detailed functional analysis in vitro, using all known possible phenylpropanoid pathway intermediates (p-coumaric, caffeic, ferulic, 5-hydroxyferulic and sinapic acids), as well as cinnamic acid. Of the 11 recombinant proteins so obtained, four were catalytically active in vitro, with fairly broad substrate specificities, confirming that the 4CL gene family in Arabidopsis has only four members. This finding is in agreement with our previous phylogenetic analyses, and again illustrates the need for comprehensive characterization of all putative 4CLs, rather than piecemeal analysis of selected gene members. All 11 proteins were expressed with a C-terminal His6-tag and functionally characterized, with one, At4CL1, expressed in native form for kinetic property comparisons. Of the 11 putative His6-tagged 4CLs, isoform At4CL1 best utilized p-coumaric, caffeic, ferulic and 5-hydroxyferulic acids as substrates, whereas At4CL2 readily transformed p-coumaric and caffeic acids into the corresponding CoA esters, while ferulic and 5-hydroxyferulic acids were converted quite poorly. At4CL3 also displayed broad substrate specificity efficiently converting p-coumaric, caffeic and ferulic acids into their CoA esters, whereas 5-hydroxyferulic acid was not as effectively utilized. By contrast, while At4CL5 is the only isoform capable of ligating sinapic acid, the two preferred substrates were 5-hydroxyferulic and caffeic acids. Indeed, both At4CL1 and At4CL5 most effectively utilized 5-hydroxyferulic acid with kenz approximately 10-fold higher than that for At4CL2 and At4CL3. The remaining seven 4CL-like homologues had no measurable catalytic activity (at approximately 100 microg protein concentrations), again bringing into sharp focus both the advantages to, and the limitations of, current database annotations, and the need to unambiguously demonstrate true enzyme function. Lastly, although At4CL5 is able to convert both 5-hydroxyferulic and sinapic acids into the corresponding CoA esters, the physiological significance of the latter observation in vitro was in question, i.e. particularly since other 4CL isoforms can effectively convert 5-hydroxyferulic acid into 5-hydroxyferuloyl CoA. Hence, homozygous lines containing T-DNA or enhancer trap inserts (knockouts) for 4cl5 were selected by screening, with Arabidopsis stem sections from each mutant line subjected to detailed analyses for both lignin monomeric compositions and contents, and sinapate/sinapyl alcohol derivative formation, at different stages of growth and development until maturation. The data so obtained revealed that this "knockout" had no significant effect on either lignin content or monomeric composition, or on the accumulation of sinapate/sinapyl alcohol derivatives. The results from the present study indicate that formation of syringyl lignins and sinapate/sinapyl alcohol derivatives result primarily from methylation of 5-hydroxyferuloyl CoA or derivatives thereof rather than sinapic acid ligation. That is, no specific physiological role for At4CL5 in direct sinapic acid CoA ligation could be identified. How the putative overlapping 4CL metabolic networks are in fact organized in planta at various stages of growth and development will be the subject of future inquiry.

Ultrastructural changes of neuronal mitochondria after transient and permanent cerebral ischemia.

BACKGROUND AND PURPOSE: Mitochondrial swelling is one of the most striking and initial ultrastructural changes after acute brain ischemia. The purpose of the present study was to examine the role of reperfusion of the cerebral cortex after transient focal cerebral ischemia on neuronal mitochondrial damage. METHODS: Male Sprague-Dawley rats (n=16) were subjected to either temporary or permanent occlusion of the middle cerebral artery and bilateral carotid arteries. Three experimental conditions were compared: group I, permanent ischemia (3, 5, and 24 hours); group II, transient ischemia (2, 24 hours of reperfusion); and sham surgery. Anesthetized rats were killed by cardiac perfusion, and brain tissue was removed ipsilaterally and contralaterally from the ischemic core section of the frontoparietal cortex. Fixed tissue was prepared for electron microscopic examination, and electron microscopic thin sections of random neurons were photographed. Perinuclear neuronal mitochondria were analyzed in a blinded manner for qualitative ultrastructural changes (compared with sham control) by 2 independent investigators using an objective grading system. RESULTS: Cortical neuronal mitochondria exposed to severe ischemic/reperfusion conditions demonstrated dramatic signs of injury in the form of condensation, increased matrix density, and deposits of electron-dense material followed by disintegration by 24 hours. In contrast, mitochondria exposed to an equivalent time of permanent ischemia demonstrated increasing loss of matrix density with pronounced swelling followed by retention of their shape by 24 hours. CONCLUSIONS: Neuronal mitochondria undergoing transient versus permanent ischemia exhibit significantly different patterns of injury. Structural damage to neuronal mitochondria of the neocortex occurs more acutely and to a greater extent during the reperfusion phase in comparison to ischemic conditions alone. Further research is in progress to delineate the role of oxygen free radical production in the observed mitochondrial damage during postischemic reoxygenation.

Placement of 19F into the center of GB1: effects on structure and stability.

A structural and thermodynamic characterization of 5F-Trp-substituted immunoglobulin binding domain B1 of streptococcal protein G (GB1) was carried out by nuclear magnetic resonance and circular dichroism spectroscopy. A single fluorine reporter atom was positioned at the center of the three-dimensional structure, uniquely poised to be exploited for studying interior properties of this protein. We demonstrate that the introduction of 5F-Trp does not affect the global and local architecture of GB1 and has no influence on the thermodynamic stability. The favorable properties of the fluorinated GB1 render this molecule a desirable model system for the development of spectroscopic methodology and theoretical calculations.

[13C]-Specific labeling of 8-2' linked (-)-cis-blechnic, (-)-trans-blechnic and (-)-brainic acids in the fern Blechnum spicant.

In vivo administration experiments using stable (13C) and radio (14C) labeled precursors established that the optically active 8-2' linked lignans, (-)-cis-blechnic, (-)-trans-blechnic and (-)-trans-brainic acids, were directly derived from L-phenylalanine, cinnamate, and p-coumarate but not either from tyrosine or acetate. The radiochemical time course data suggest that the initial coupling product is (-)-cis-blechnic acid, which is then apparently converted into both (-)-trans-blechnic and (-)-trans-brainic acids in vivo. These findings provide additional evidence for vascular plant proteins engendering distinct but specific phenolic radical-radical coupling modes, i.e., for full control over phenylpropanoid coupling in vivo, whether stereoselective or regiospecific.

Quantification of wheat straw lignin structure by comprehensive NMR analysis.

A further understanding of the structure of lignin from herbaceous crops is needed for advancing technologies of lignocellulosic biomass processing and utilization. A method was established in this study for analyzing structural motifs found in milled straw lignin (MSL) and cellulase-digested lignin (CEL) isolated from wheat straw by combining quantitative (13)C and HSQC NMR spectral analyses. The results showed that guaiacyl (G) was the predominant unit in wheat straw cell wall lignin over syringyl (S) and hydroxyphenyl (H) units. Up to 8.0 units of tricin were also detected in wheat straw lignin per 100 aromatic rings. Various interunit linkages, including ß-O-4, ß-5, ß-ß', ß-1, α, ß-diaryl ether, and 5-5'/4-O-ß' as well as potential lignin-carbohydrate complex (LCC) bonds, were identified and quantified. These findings provide useful information for the development of biofuels and lignin-based materials.