In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - III. Comparison of enzymes derived from psychrophilic, mesophilic or thermophilic sources

A completely randomised study was completed to examine the influence of fibrolytic enzymes derived from psychrophilic, (F), mesophilic, (L) or thermophilic (Ta) sources, applied at ensiling, on the chemical characteristics and in vitro rumen fermentation of maize silage, assessed using the Reading Pressure Technique (RPT). Treatments, all in triplicate, consisted of untreated maize forage or treated with preparations F, L, Ta or a mixture (1: 1, v/v) of F and L (FL), at two levels each, and ensiled for 210 days in plastic mini-silos. Addition of enzymes L decreased (P < 0.05) silage pH relative to the control, whereas enzyme Ta tended (P < 0.10) to reduce it. Preparations F, L and Ta tended to reduce (P < 0.10) the fibre contents of the silages, with effects being attributable to a decrease in the cellulose fraction. Starch contents were reduced (P < 0.05) in the treatments including enzyme F. End-point (96 h) gas production (GP) values did not differ among treatments, suggesting that enzymes did not change the total amount of fermentable substrate. However, consistent with the decrease in starch contents, adding enzyme F reduced (P < 0.05) GP at most incubation times. Addition of enzymes increased (P < 0.05) the initial (6 h) organic matter degradation (OMD) levels in all but one treatment (F), with increases of 14, 19, and 26% for preparations L, Ta, and FL, respectively, averaged across levels. Furthermore, the addition of enzymes increased (P < 0.05) the soluble OM losses, however, these increases did not fully account for the initial increase in OMD. The latter suggests that enzymes increased solubility and also altered silage structure, making it more amenable to degradation by ruminal microorganisms. As a result of the increase in OMD, without a concomitant increase in GP, the fermentation efficiency was greatly increased (P < 0.05) in enzyme treatments. Addition of enzymes to maize at ensiling, particularly those from the mesophilic and thermophilic sources used here, have the potential to increase the initial rate of silage OMD. (C) 2003 Elsevier B.V. All rights reserved.

Effect of feeding supplemental fibrolytic enzymes or soluble sugars with malic acid on milk production

Two trials were conducted to evaluate effects of feeding supplemental fibrolytic enzymes or soluble sugars and malic acid on milk production. In trial 1, 257 cows at four sites were fed a basal diet consisting of no more than 60% of forage DM as corn silage and less than 40% as alfalfa hay. Cows were assigned randomly within site, parity, and two stages of lactation to: 1) control; 2) enzyme A; 3) enzyme B; and 4) soluble sugars and malic acid. There was a 14-d pretreatment and an 84-d treatment period. Enzyme solutions were sprayed on either the forage component or the TMR each day while mixing feed. Trial 2 was similar, except 122 cows at one site in the United Kingdom were fed diets containing forage that was 75% corn silage and 25% grass silage, and all cows began the study between 25 to 31 DIM. Mean milk productions for 233 cows that completed trial 1 were 32.9, 32.5, 32.4, and 32.9 kg/d for control, enzyme A, enzyme B, and soluble sugars and malic acid, respectively. Mean milk productions for 116 cows that completed trial 2 were 28.2, 27.9, 28.8, and 28.4 kg/d, respectively. In vitro analyses of the activities of enzyme solutions indicated that all major cellulose and hemicellulose degrading activities were present; however, the pH optima (approximate pH = 4 to 5) were more acidic, and the temperature optimum (approximately 50 C) was greater than normal pH and temperature in the rumen. If fibrolytic activity in the rumen is a major mechanism of action of supplemental fibrolytic enzymes, it appears that considerable activity of these preparations was lost due to conditions in the rumen. In conclusion, feeding supplemental fibrolytic enzymes or malic acid with soluble sugars had no effect on milk production under the conditions used in this study.

The cell wall and secretory proteome of a tobacco cell line synthesising secondary wall

The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion-exchange chromatography, could be determined accurately since, xylem-specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available.

Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its consequences for cellulose extractability

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490 bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its conscequence for cellulose extractability

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490 bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

Novel high-resolution characterization of ancient DNA reveals C > U-type base modification events as the sole cause of post mortem miscoding lesions

Ancient DNA (aDNA) research has long depended on the power of PCR to amplify trace amounts of surviving genetic material from preserved specimens. While PCR permits specific loci to be targeted and amplified, in many ways it can be intrinsically unsuited to damaged and degraded aDNA templates. PCR amplification of aDNA can produce highly-skewed distributions with significant contributions from miscoding lesion damage and non-authentic sequence artefacts. As traditional PCR-based approaches have been unable to fully resolve the molecular nature of aDNA damage over many years, we have developed a novel single primer extension (SPEX)-based approach to generate more accurate sequence information. SPEX targets selected template strands at defined loci and can generate a quantifiable redundancy of coverage; providing new insights into the molecular nature of aDNA damage and fragmentation. SPEX sequence data reveals inherent limitations in both traditional and metagenomic PCR-based approaches to aDNA, which can make current damage analyses and correct genotyping of ancient specimens problematic. In contrast to previous aDNA studies, SPEX provides strong quantitative evidence that C U-type base modifications are the sole cause of authentic endogenous damage-derived miscoding lesions. This new approach could allow ancient specimens to be genotyped with unprecedented accuracy.

Identification of a novel mammalian post-translational modification, phosphocholine, on placental secretory polypeptides

Placental neurokinin B appears to be post-translationally modified by phosphocholine (PC) attached to the aspartyl side chain at residue 4 of the mature peptide. Corticotrophin releasing factor (CRF) was found to be expressed by the rat placenta with the main secreted forms being phosphocholinated proCRF+/- one or two polysaccharide moieties. A combination of high-pressure liquid chromatography (HPLC) and two-site immunometric analysis suggested that PC was also attached to the placental precursors of adrenocorticotrophin, hemokinin, activin and follistatin. However, the fully processed forms of rat placental activin and CRF were free of PC. Formerly, the parasitic filarial nematodes have used PC as a post-translational modification, attached via the polysaccharicle moiety of certain secretory glycoproteins to attenuate the host immune system allowing parasite survival, but it is the PC group itself which endows the carrier with the biological activity. The fact that treatment of proCRF peptides with phospholipase C but not endoglycosidase destroyed PC immunoreactivity suggested a simpler mode of attachment of PC to placental peptides than that used by nematodes. Thus, it is possible that by analogy the placenta uses its secreted phosphocholinated hormones to modulate the mother's immune system and help protect the placenta from rejection.

Cupins: the most functionally diverse protein superfamily?

The cupin superfamily of proteins, named on the basis of a conserved β-barrel fold (‘cupa’ is the Latin term for a small barrel), was originally discovered using a conserved motif found within germin and germin-like proteins from higher plants. Previous analysis of cupins had identified some 18 different functional classes that range from single-domain bacterial enzymes such as isomerases and epimerases involved in the modification of cell wall carbohydrates, through to two-domain bicupins such as the desiccation-tolerant seed storage globulins, and multidomain transcription factors including one linked to the nodulation response in legumes. Recent advances in comparative genomics, and the resolution of many more 3-D structures have now revealed that the largest subset of the cupin superfamily is the 2-oxyglutarate-Fe2+ dependent dioxygenases. The substrates for this subclass of enzyme are many and varied and in total amount to probably 50–100 different biochemical reactions, including several involved in plant growth and development. Although the majority of enzymatic cupins contain iron as an active site metal, other members contain either copper, zinc, cobalt, nickel or manganese ions as a cofactor, with each cofactor allowing a different type of chemistry to occur within the conserved tertiary structure. This review discusses the range of structures and functions found in this most diverse of superfamilies.

Fragrance release from the surface of branched poly(amide)s

Enzymes are powerful tools in organic synthesis that are able to catalyse a wide variety of selective chemical transformations under mild and environmentally friendly conditions. Enzymes such as the lipases have also found applications in the synthesis and degradation of polymeric materials. However, the use of these natural catalysts in the synthesis and the post-synthetic modification of dendrimers and hyperbranched molecules is an application of chemistry yet to be explored extensively. In this study the use of two hydrolytic enzymes, a lipase from Candida cylindracea and a cutinase from Fusarium solani pisii, were investigated in the selective cleavage of ester groups situated on the peripheral layer of two families of branched polyamides. These branched polyamides were conjugated to simple fragrances citronellol and L-menthol via ester linkages. Hydrolysis of the ester linkage between the fragrances and the branched polyamide support was carried out in aqueous buffered systems at slightly basic pH values under the optimum operative conditions for the enzymes used. These preliminary qualitative investigations revealed that partial cleavage of the ester functionalities from the branched polyamide support had occurred. However, the ability of the enzymes to interact with the substrates decreased considerably as the branching density, the rigidity of the structure and the bulkiness of the polyamide-fragrance conjugates increased.

Surface modification of nylon 6,6 using a carbene insertion approach

The diazirine functionalised fluorenone, 3-[3-(trifluoromethyl)diazirin-3-yl]phenyl-9-oxo-9H-fluorene-2-carboxyla te was synthesised to act as a model compound capable of modifying a wide variety of polymeric substrates. Photochemical activation of the diazirine moiety of the fluorenone derivative was utilised to afford highly reactive carbenes capable of insertion into or addition to a wide variety of functionalities. In this paper the photoinduced attachment of a fluorenone derivative to nylon 6,6 has been studied using UV-visible spectroscopic analysis. Incorporation of the fluorenone chromophore onto the backbone of nylon at different loading levels and after different coating cycles has been investigated and is detailed in this paper.

Mechanical effects of plant cell wall enzymes on xyloglucan/cellulose composites

Xyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties. In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan). Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris. Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase). Both 'hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction. Consequent major changes in cellulose fibril architecture were observed. In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility. On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio. No change in composite stiffness or extensibility resulted, but a significant increase in creep behaviour was observed in the presence of active tXET. These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-specific enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes. Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin.

Effect of oxidized low-density lipoprotein on differential gene expression in primary human endothelial cells

Oxidative modification of low-density lipoprotein (LDL) plays an important role in the initiation and progression of atherosclerosis. It has been proposed that the biological action of oxidized LDL (ox-LDL) may be partially attributed to its effect on a shift of the pattern of gene expression in endothelial cells. To examine the transcriptional response to ox-LDL, we applied cDNA array technology to cultured primary human endothelial cells challenged with oxidized human LDL. A twofold or greater difference in the expression of a particular gene was considered a significant difference in transcript abundance. Seventy-eight of the 588 genes analyzed were differentially expressed in response to the treatment. Ox-LDL significantly affected the expression of genes encoding for transcription factors, cell receptors, growth factors, adhesion molecules, extracellular matrix proteins, and enzymes involved in cholesterol metabolism. The alteration of the expression pattern of several genes was substantiated post hoc using RT-PCR. The experimental strategy identified several novel ox-LDL-sensitive genes associated with a "response to injury" providing a conceptual background to be utilized for future studies addressing the molecular basis of the early stages of atherogenesis.

Localisation studies of cell-wall degrading enzymes in soybean

Postembedding immunoelectron microscopy has been used to investigate the diffusibility of an endo-beta-1,4-glucanase and a xylanase from A. niger in soybean. The results showed more specific localisation of the enzymes into the protein and lipid bodies of soybean cells. This was against our hypothesis that suggested that the enzymes should be localised in the cell wall.

Modulation of detoxification enzymes by watercress: in vitro and in vivo investigations in human peripheral blood cells

Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes. The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner. In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1-10 mu L/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1-10 mu M). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes. In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 mu L/mL WCE, 3.7-fold 1 mu M PEITC) and SOD2 (12.1-fold, 10 mu L/mL WCE, 7.3-fold, 10 mu M PEITC), and increased SOD2 activity (1.9-fold, 10 mu L/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact. The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.

Plant- and marine-derived n-3 polyunsaturated fatty acids have differential effects on fasting and postprandial blood lipid concentrations and on the susceptibility of LDL to oxidative modification in moderately hyperlipidemic subjects

Background: Dietary a-linolenic acid (ALA) can be converted to long-chain n-3 polyunsaturated fatty acids (PUFAs) in humans and may reproduce some of the beneficial effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on cardiovascular disease risk factors. Objective: This study aimed to compare the effects of increased dietary intakes of ALA and EPA+DHA on a range of atherogenic risk factors. Design: This was a placebo-controlled, parallel study involving 150 moderately hyperlipidemic subjects randomly assigned to 1 of 5 interventions: 0.8 or 1.7 g EPA+DHA/d, 4.5 or 9.5 g ALA/d, or an n-6 PUFA control for 6 mo. Fatty acids were incorporated into 25 g of fat spread and 3 capsules to be consumed daily. Results: The change in fasting or postprandial lipid, glucose, or insulin concentrations or in blood pressure was not significantly different after any of the n-3 PUFA interventions compared with the n-6 PUFA control. The mean (+/-SEM) change in fasting triacylglycerols after the 1.7-g/d EPA+DHA intervention (-7.7 +/- 4.99%) was significantly (P < 0.05) different from the change after the 9.5-g/d ALA intervention (10.9 +/- 4.5%). The ex vivo susceptibility of LDL to oxidation was higher after the 1.7-g/d EPA+DHA intervention than after the control and ALA interventions (P < 0.05). There was no significant change in plasma a-tocopherol concentrations or in whole plasma antioxidant status in any of the groups. Conclusion: At estimated biologically equivalent intakes, dietary ALA and EPA+DHA have different physiologic effects.