Biomedical subjects

R D Hatfield

Publications and source records attributed to R D Hatfield.

16 recordsLinked to original sources

PubMed2001

Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR.

Studying lignin-biosynthetic-pathway mutants and transgenics provides insights into plant responses to perturbations of the lignification system, and enhances our understanding of normal lignification. When enzymes late in the pathway are downregulated, significant changes in the composition and structure of lignin may result. NMR spectroscopy provides powerful diagnostic tools for elucidating structures in the difficult lignin polymer, hinting at the chemical and biochemical changes that have occurred. COMT (caffeic acid O-methyl transferase) downregulation in poplar results in the incorporation of 5-hydroxyconiferyl alcohol into lignins via typical radical coupling reactions, but post-coupling quinone methide internal trapping reactions produce novel benzodioxane units in the lignin. CAD (cinnamyl alcohol dehydrogenase) downregulation results in the incorporation of the hydroxycinnamyl aldehyde monolignol precursors intimately into the polymer. Sinapyl aldehyde cross-couples 8-O-4 with both guaiacyl and syringyl units in the growing polymer, whereas coniferyl aldehyde cross-couples 8-O-4 only with syringyl units, reflecting simple chemical cross-coupling propensities. The incorporation of hydroxycinnamyl aldehyde and 5-hydroxyconiferyl alcohol monomers indicates that these monolignol intermediates are secreted to the cell wall for lignification. The recognition that novel units can incorporate into lignins portends significantly expanded opportunities for engineering the composition and consequent properties of lignin for improved utilization of valuable plant resources.

Alcohol Oxidoreductases↗

PubMed2001

Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method.

Lignin extracted with acidic dioxane was investigated as a possible standard for quantitatively determining lignin content in plant samples using the spectrophotometric method employing acetyl bromide. Acidic dioxane lignins were analyzed for carbohydrate, total protein, nitrobenzene oxidation products, and UV spectral characteristics. Total carbohydrate content of isolated lignins ranged from 2.21 to 5.70%, while protein ranged from 0.95 to 6.06% depending upon the plant source of the original cell wall sample. Nitrobenzene analysis indicated differences in the amount of guaiacyl and syringyl units making up the lignins, but this did not alter the UV spectrum of lignin solubilized in acetyl bromide. Regression equations developed for the acetyl bromide method using the isolated lignins for all the plant samples were similar to each other. Lignin values obtained by the acetyl bromide method were similar to the lignin values obtained as acid insoluble residues following a Klason lignin procedure.

Acetates↗

PubMed2001

Excision and repair of the peripheral ossifying fibroma: a report of 3 cases.

BACKGROUND: The peripheral ossifying fibroma (POF), one of the most common gingival lesions, has a recurrence rate of nearly 20%. To minimize the reappearance of this lesion, it must be completely excised. In the maxillary anterior region, total excision of a POF can result in an unsightly gingival defect. METHODS: Three cases are presented in which a POF was excised from the gingiva facial to a maxillary central incisor. One of these lesions had previously undergone 2 cycles of conservative excision and recurrence. In all cases, the lesions were excised down to bone. Each of the resulting gingival defects was repaired by a distinct plastic surgery procedure, including a laterally positioned flap, a subepithelial connective tissue graft, and a coronally positioned flap. RESULTS: The defects resulting from the biopsies were satisfactorily repaired. The patients were followed over postsurgical intervals of 10 to 30 months. None of the lesions recurred. CONCLUSIONS: It is customary to manage POF by aggressive excisional biopsy. Several different surgical approaches may potentially be used to repair the resultant gingival defect and minimize patient esthetic concerns.

Adult↗

PubMed2000

Cross-linking of maize walls by ferulate dimerization and incorporation into lignin.

Cross-linking of xylans and lignin by ferulates was investigated with primary maize walls acylated with 2% ferulate and with ferulate ethyl esters. Peroxidase-mediated coupling of wall ferulate and ethyl ferulate yielded mostly 8-coupled products, including three new dehydrodimers. Significant quantities of 5-5-coupled diferulate formed only within walls, suggesting that matrix effects influence dimer formation. Over 60% of wall ferulate dimerized upon H2O2 addition, suggesting that xylan feruloylation is highly regulated during wall biosynthesis to permit extensive dimer formation at the onset of lignification. During lignification, ferulate and 5-5-coupled diferulate copolymerized more rapidly and formed fewer ether-linked structures with coniferyl alcohol than 8-5-, 8-O-4-, and 8-8-coupled diferulates. The potential incorporation of most ferulates and diferulates into lignin exceeded 90%. As a result, xylans become extensively cross-linked by ferulate dimerization and incorporation to lignin, but only a small and variable proportion of these cross-links is measurable by solvolysis of lignified walls.

Cell Wall↗

PubMed2000

Fermentation of a bacterial cellulose/xylan composite by mixed ruminal microflora: implications for the role of polysaccharide matrix interactions in plant cell wall biodegradability.

Growth of the cellulose-synthesizing bacterium Acetobacter xylinum ATCC 53524 in media supplemented with 5% (w/v) glucose and 0.2% (w/v) of a water-soluble, nearly linear xylan from tobacco stalks resulted in the synthesis of a highly crystalline composite having a xylose/glucose ratio ranging from 0.06 to 0.24. The digestion of one composite (88% cellulose/12% xylan) by mixed ruminal microflora displayed kinetics of gas production similar to those of an unassociated mixture of the two components added in a xylan/cellulose ratio similar to that of the composite. The data suggest that intimate association of xylan and cellulose, as is typically found in secondary plant cell walls, does not inhibit the rate of digestion of the component polysaccharides.

Acetobacter↗

PubMed1999

NMR characterization of lignins in Arabidopsis altered in the activity of ferulate 5-hydroxylase.

Nuclear magnetic resonance (NMR) of isolated lignins from an Arabidopsis mutant deficient in ferulate 5-hydroxylase (F5H) and transgenic plants derived from the mutant by overexpressing the F5H gene has provided detailed insight into the compositional and structural differences between these lignins. Wild-type Arabidopsis has a guaiacyl-rich, syringyl-guaiacyl lignin typical of other dicots, with prominent beta-aryl ether (beta-O-4), phenylcoumaran (beta-5), resinol (beta-beta), biphenyl/dibenzodioxocin (5-5), and cinnamyl alcohol end-group structures. The lignin isolated from the F5H-deficient fah1-2 mutant contained only traces of syringyl units and consequently enhanced phenylcoumaran and dibenzodioxocin levels. In fah1-2 transgenics in which the F5H gene was overexpressed under the control of the cauliflower mosaic virus 35S promoter, a guaiacyl-rich, syringyl/guaiacyl lignin similar to the wild type was produced. In contrast, the isolated lignin from the fah1-2 transgenics in which F5H expression was driven by the cinnamate 4-hydroxylase promoter was almost entirely syringyl in nature. This simple lignin contained predominantly beta-aryl ether units, mainly with erythro-stereochemistry, with some resinol structures. No phenylcoumaran or dibenzodioxocin structures (which require guaiacyl units) were detectable. The overexpression of syringyl units in this transgenic resulted in a lignin with a higher syringyl content than that in any other plant we have seen reported.

Arabidopsis↗

PubMed1999

Unexpected variation in lignin.

Recent studies on mutant and transgenic plants indicate that lignification may be far more flexible than previously realized. Pines with a mutation affecting the biosynthesis of the major lignin precursor, coniferyl alcohol, show a high level of an unusual subunit, dihydroconiferyl alcohol. These results argue in favor of an increased potential for genetic modification of lignin and indicate that our knowledge of the biosynthesis of lignin is far from complete.

Alcohol Oxidoreductases↗

PubMed1999

Using the acetyl bromide assay to determine lignin concentrations in herbaceous plants: some cautionary notes.

The acetyl bromide assay was developed to provide a rapid and sensitive method for quantifying lignin in woody plant species. The original procedure cautioned against prolonged reaction times and advised keeping the reaction temperature at 70 degrees C to prevent excessive carbohydrate degradation that would skew the absorption spectra. Characterization of the reaction conditions revealed that the acetyl bromide reagent readily degrades xylans, a prominent polysaccharide group within all lignified plants. This degradation results in increased absorbance in the 270-280 nm region that is used to quantify lignin. The degradation of xylans is temperature dependent and is exacerbated by the addition of perchloric acid. Lowering the reaction temperature to 50 degrees C and increasing the reaction time from 2 to 4 h allows complete lignin solubilization but minimizes degradation of the xylans.

Acetates↗

PubMed1999

Are lignins optically active?

The accepted derivation of lignins from non(enzymatically)-controlled radical coupling reactions has been recently challenged, and it is relevant to ascertain unequivocally whether lignins are or are not (as normally assumed) optically active. Two approaches were used. First, DFRC (derivatization followed by reductive cleavage) dimers derived from beta-5- and beta-beta-units in pine lignins, which certainly retain unaltered chiral centers (as well as beta-1- and beta-O-4-units where the intactness may be debated), were shown to be optically inactive by circular dichroism (CD) and chiral high-performance liquid chromatography. CD of beta-5-derived dimers following enantiomeric separation readily demonstrated the sensitivity of the method. Second, no optical activity could be detected (above 250 nm to avoid carbohydrate contributions) by CD of lignin isolates from pine, kenaf, maize, or a CAD-deficient pine mutant. Representative lignins are therefore not, within limits of detection by these methods, optically active.

Carbohydrate Conformation↗

PubMed1999

Comparison of Tifton 85 and Coastal bermudagrasses for yield, nutrient traits, intake, and digestion by growing beef steers.

A study was undertaken to compare Tifton 85 (T85) and Coastal (CBG) bermudagrasses for effects of cultivar and age at harvest on yields of DM and digestible DM, in vitro digestion, nutrient content, cell wall composition, in situ digestion kinetics, and feed intake and digestion by growing beef steers. In Exp. 1, T85 and CBG forages staged for growth in May or July of 1993 were harvested at 3, 4, 5, 6, 7, and 8 wk from subplots. Tifton 85 bermudagrass had 7.1% greater DM yield, 18.2% higher (P < .05) digestible DM yield, and 7.1% greater IVDMD than CBG, and, after 5 wk of forage growth, IVDMD of both T85 and CBG decreased with increased age at harvest (P < .05). In Exp. 2, T85 and CBG forages staged for growth in July 1997 were harvested at 2, 3, 4, 5, 6, and 7 wk from subplots. Even though T85 had higher concentrations of NDF and ADF than CBG, T85 had 34.1% higher DM yield, 47.9% higher digestible DM, 55.0% higher digestible NDF, 91.7% higher digestible ADF, greater IVDMD, in vitro NDF and ADF disappearances, and higher in situ DM and NDF digestion (P < .05). Coastal bermudagrass had higher concentrations of lignin and lower concentrations of total neutral sugars, arabinose, glucose, and xylose than T85 (P < .05). In vitro digestibilities of DM, NDF, and ADF were lower and concentrations of ADF and lignin were greater for 7- vs 6-wk harvests of both T85 and CBG (P < .05). In Exp. 3, T85 and CBG forages staged for growth in July 1997 were harvested as hay at 3, 5, and 7 wk from .8-ha pastures and fed to 36 individually penned growing beef steers (initial BW = 244 kg) to quantify ad libitum intake without supplementation. Tifton 85 bermudagrass had lower concentrations of lignin and ether-linked ferulic acid and greater concentrations of NDF, ADF, hemicellulose, and cellulose than CBG (P < .05). Steers fed T85 had higher (P < .05) digestion of DM, OM, NDF, ADF, hemicellulose, and cellulose than steers fed CBG. Digestion of NDF, ADF, hemicellulose, and cellulose decreased (P < .05) with increased age at harvest for both cultivars. In conclusion, T85 produced more DM and had more digestible nutrients in vitro, in situ, and in vivo than CBG, and 3 and 5 wk of growth would be recommended ages to harvest either cultivar.

Animal Nutritional Physiological Phenomena↗

PubMed1998

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase.

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl-SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl-SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Journal Article↗

PubMed1997

Abnormal lignin in a loblolly pine mutant.

Novel lignin is formed in a mutant loblolly pine (Pinus taeda L.) severely depleted in cinnamyl alcohol dehydrogenase (E.C. 1.1.1.195), which converts coniferaldehyde to coniferyl alcohol, the primary lignin precursor in pines. Dihydroconiferyl alcohol, a monomer not normally associated with the lignin biosynthetic pathway, is the major component of the mutant's lignin, accounting for approximately 30 percent (versus approximately 3 percent in normal pine) of the units. The level of aldehydes, including new 2-methoxybenzaldehydes, is also increased. The mutant pines grew normally indicating that, even within a species, extensive variations in lignin composition need not disrupt the essential functions of lignin.

Alcohol Oxidoreductases↗

PubMed1997

Excretion of purine derivatives by Holstein cows abomasally infused with incremental amounts of purines.

Five multiparous, ruminally cannulated Holstein cows (two lactating and three dry) weighing (X +/- SD) 667 +/- 35 kg were used to study the effect of abomasal purine infusion on the excretion of purine derivatives. Cows were fed corn silage four times daily at 90% of ad libitum intake (X = 9.16 kg of dry matter/d). Purines were infused into the abomasum as brewer's yeast suspensions in five incremental amounts (0 to 380 mmol/d) during five experimental periods according to a 5 x 5 Latin square design. Periods were 7 d; purine infusions were conducted during the last 4 d, and urine was collected during the last 3 d of each period. Ruminal purine outflow in all cows was measured during an experimental period immediately preceding and immediately following the five infusion periods and in each cow during the 0-mmol/d infusion period of the experiment. The relationship between total (milk plus urine) daily excretion of purine derivatives (allantoin plus uric acid) and total (abomasal infusion plus ruminal outflow) daily purine flow was quantified by linear regression analysis and was described by the relationship: Y = 0.856X + 103 (r2 = 0.93). The slope (0.856) indicated that 86% of purines that reached the omasum were excreted as purine derivatives. In the two lactating cows, urinary purine derivatives accounted for 98.4% of the total purine derivatives that were excreted. Ruminal flow of microbial CP can be estimated from the CP:purine ratio of ruminal microorganisms and the excretion of purine derivatives.

Abomasum↗

PubMed1993

Inhibition of ruminal cellulose fermentation by extracts of the perennial legume cicer milkvetch (Astragalus cicer).

Cicer milkvetch (Astragalus cicer L.) is a perennial legume used as a pasture or rangeland plant for ruminants. A study was undertaken to determine whether reported variations in its ruminal digestibility may be related to the presence of an antinutritive material. In vitro fermentation of neutral detergent fiber (NDF) of cicer milkvetch by mixed rumen microflora was poorer than was the fermentation of NDF in alfalfa (Medicago sativa L.). Fermentation of cicer milkvetch NDF was improved by preextraction of the ground herbage with water for 3 h at 39 degrees C. Such water extracts selectively inhibited in vitro fermentation of pure cellulose by mixed ruminal microflora and by pure cultures of the ruminal bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85. Inhibition of the cellulose fermentation by mixed ruminal microflora was dependent upon the concentration of cicer milkvetch extract and was overcome upon prolonged incubation. Pure cultures exposed to the extract did not recover from inhibition, even after long incubation times, unless the inhibitory agent was removed (viz., by dilution of inhibited cultures into fresh medium). The extract did not affect the fermentation of cellobiose by R. flavefaciens but did cause some inhibition of cellobiose fermentation by F. succinogenes. Moreover, the extracts did not inhibit hydrolysis of crystalline cellulose, carboxymethyl cellulose, or p-nitrophenylcellobioside by supernatants of these pure cultures of cellulolytic bacteria or by a commercial cellulase preparation from the fungus Trichoderma reesei. The agent caused cellulose-adherent cells to detach from cellulose fibers, suggesting that the agent may act, at least in part, by disrupting the glycocalyx necessary for adherence to, and rapid digestion of, cellulose.

Animals↗

PubMed1992

Effect of white rot basidiomycetes on chemical composition and in vitro digestibility of oat straw and alfalfa stems.

Five white rot basidiomycetes were evaluated for their potential to improve ruminal degradation of oat straw and alfalfa stems. Phanerochaete chrysosporium (PC), Scytinostroma galactinum (SG), Phlebia tremellosa (PT), Phellinus pini (PP), and Pholiota mutabilis (PM) were incubated on oat straw and alfalfa stems for 30 d at 28 degrees C and 90% relative humidity. Detergent fiber and total fiber components (neutral sugars, uronic acids, Klason lignin [KL], and ester- and ether-linked non-core lignin phenolics), core lignin nitrobenzene oxidation products, and IVDMD were determined. Electron microscopy of KMnO4-stained and cellulase/colloidal gold-labeled sections was used to monitor fungal activity. Large losses of DM were noted for all fungal species on both substrates. Lignin (KL and ADL) was removed (P less than .05) from oat straw by PC and PT treatment, but no net loss of lignin was observed for fungal treatment of alfalfa stems. Cell-wall polysaccharides were removed from both substrates by fungal activity. Only PC increased (P less than .05) IVDMD of oat straw, and SG, PT, PP, and PC treatment decreased (P less than .05) IVDMD of alfalfa stems, presumably because the fungi removed the most readily fermentable polysaccharides. Transmission electron microscopy using KMnO4 staining showed a nonselective white rot attack. Cytochemical studies using colloidal gold-labeled exo- and endocellulases were used to map the location of cellulose in the cell wall before and after decay by the white rot fungi. All the white rot fungi tested had eroded and thinned cell walls. Residual cell walls were well-labeled; both endo- and exocellulose-colloidal gold identified the cellulosic wall material that remained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Feed↗

PubMed1991

Synthesis of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and its use as a substrate to assess feruloyl esterase activity.

A synthetic scheme was developed for the production of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside (FA-Ara) in gram quantities. This molecule accurately models the chemical attachment of ferulic acid to polysaccharides found in cell walls of plants in the Gramineae family. It is therefore a realistic substrate that can be used to monitor feruloyl esterase activity. Ultraviolet spectral analysis indicated that FA-Ara has an absorption maximum distinct from the hydrolytic product, ferulic acid (FA), over a wide range of solution pH values. The log molar extinction coefficient ranges from 4.16 to 4.36 for FA-Ara and 4.16 to 4.33 for FA depending upon the pH of the buffered solution. Consequently a convenient spectrophotometric assay can be utilized to monitor esterase activity. Three different methods were developed for using this model substrate to assess esterase activity, including thin-layer chromatography, a spectrophotometric assay, and the use of high-performance liquid chromatography.

Arabinose↗