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 effect of acetylsalicylic acid and oxalic acid on Myzus persicae and Aphidius colemani

Plants can respond to damage by pests with both induced direct defences and indirect defences by the attraction of their natural enemies. Foliar application of several plant-derived chemicals, such as salicylic acid and oxalic acid, can induce these defence mechanisms. The effect of acetylsalicylic acid and oxalic acid on the aphid Myzus persicae Sulzer (Homoptera: Aphididae) and its parasitoid Aphidius colemani Viereck (Hymenoptera: Aphidiidae) was investigated. Experiments were carried out with direct application of acetylsalicylic and oxalic acids on these insects, as well as choice and no-choice tests using foliar application of both chemicals on Brussels sprouts plants, Brassica oleracea var. gemmifera L. (Brassicaceae). Parasitoids were given a choice between treated and untreated plants for oviposition, and the effects of the chemicals on aphid and parasitoid development were determined. Although direct application of both chemicals increased aphid mortality, their foliar application did not induce resistance against aphids. The foliar application of such compounds, even in low concentration as shown in the choice tests, has the potential to induce indirect plant defences against aphids by encouraging aphid parasitisation. Although the direct application of both chemicals reduced parasitoid emergence from their hosts, the foliar application of acetylsalicylic acid and low concentrations of oxalic acid did not have a negative effect on parasitoid emergence ability. However, 10 mm oxalic acid reduced the number of emerged parasitoids in no-choice experiments. This study shows that foliar application of acetylsalicylic and oxalic acids has the potential to encourage aphid parasitisation, but care is needed as high concentrations of oxalic acid can have a negative effect on these beneficial organisms.

Probing the receptor interactions ofan H5 avian influenza virus using a baculovirus expression system and functionalised poly(acrylic acid) ligands

Influenza viruses attach to host cells by binding to terminal sialic acid (Neu5Ac) on glycoproteins or glycolipids. Both the linkage of Neu5Ac and the identity of other carbohydrates within the oligosaccharide are thought to play roles in restricting the host range of the virus. In this study, the receptor specificity of an H5 avian influenza virus haemagglutinin protein that has recently infected man (influenza strain A/Vietnam/1194/04) has been probed using carbohydrate functionalised poly(acrylic acid) polymers. A baculovirus expression system that allows facile and safe analysis of the Neu5Ac binding specificity of mutants of H5 HA engineered at sites that are predicted to effect a switch in host range has also been developed. (C) 2007 Elsevier Ltd. All rights reserved.

EfeUOB (YcdNOB) is a tripartite, acid-induced and CpxAR-regulated, low-pH Fe2+ transporter that is cryptic in Escherichia coli K-12 but functional in E-coli O157 : H7

Escherichia coli possesses iron transporters specific for either Fe2+ or Fe3+. Although Fe2+ is far more soluble than Fe3+, it rapidly oxidizes aerobically at pH >= 7. Thus, FeoAB, the major Fe2+ transporter of E. coli, operates anaerobically. However, Fe2+ remains stable aerobically under acidic conditions, although a low-pH Fe2+ importer has not been previously identified. Here we show that ycdNOB (efeUOB) specifies the first such transporter. efeUOB is repressed at high pH by CpxAR, and is Fe2+-Fur repressed. EfeU is homologous to the high-affinity iron permease, Ftr1p, of Saccharomyces cerevisiae and other fungi. EfeO is periplasmic with a cupredoxin N-terminal domain; EfeB is also periplasmic and is haem peroxidase-like. All three Efe proteins are required for Efe function. The efeU gene of E. coli K-12 is cryptic due to a frameshift mutation - repair of the single-base-pair deletion generates a functional EfeUOB system. In contrast, the efeUOB operon of the enterohaemorrhagic strain, O157:1147, lacks any frameshift and is functional. A 'wild-type' K-12 strain bearing a functional EfeUOB displays a major growth advantage under aerobic, low-pH, low-iron conditions when a competing metal is provided. Fe-55 transport assays confirm the ferrous iron specificity of EfeUOB.

Adventitious root formation in anacardium occidentale L. in response to phytohormones and removal of roots

Despite advances in tissue culture techniques, propagation by leafy, softwood cuttings is the preferred, practical system for vegetative reproduction of many tree and shrub species. Species are frequently defined as 'difficult'- or 'easy-to-root' when propagated by conventional cuttings. Speed of rooting is often linked with ease of propagation, and slow-to-root species may be 'difficult' precisely because tissues deteriorate prior to the formation of adventitious roots. Even when roots form, limited development of these may impair the establishment of a cutting. In this study we used softwood cuttings of cashew (Anacardium occidentale), a species considered as 'difficult-to-root'. We aimed to test the hypothesis that speed, and extent of early rooting, is critical in determining success with this species; and that the potential to form adventitious roots will decrease with time in the propagation environment. Using two genotypes, initial rooting rates were examined in the presence or absence of exogenous auxin. In cuttings that formed adventitious roots, either entire roots or root tips were removed, to determine if further root formation/development was feasible. To investigate if subsequent root responses were linked to phytohormone action, a number of cuttings were also treated with either exogenous auxin (indole-3-butyric acid-IBA) or cytokinin (zeatin). Despite the reputation of Anacardium as being 'difficult-to-root', we found high rooting rates in two genotypes (AC 10 and CCP 1001). Removing adventitious roots from cuttings and returning them to the propagation environment, resulted in subsequent re-rooting. Indeed, individual cuttings could develop new adventitious roots on four to five separate occasions over a 9 week period. Data showed that rooting potential increased, not decreased with time in the propagation environment and that cutting viability was unaffected. Root expression was faster (8-15 days) after the removal of previous roots compared to when the cuttings were first stuck (21 days). Exposing cuttings to IBA at the time of preparation, improved initial rooting in AC 10, but not in CCP 1001. Application of IBA once roots had formed had little effect on subsequent development, but zeatin reduced root length and promoted root number and dry matter accumulation. These results challenge our hypothesis, and indicate that rooting potential remains high in Anacardium. The precise mechanisms that regulate the number of adventitious roots expressed, remain to be determined. Nevertheless, results indicate that rooting potential can be high in 'difficult-to-root' species, and suggest that providing supportive environments is the key to expressing this potential. (c) 2006 Elsevier B.V. All rights reserved.

Cryopreservation of cocoa (Theobroma cacao L.) somatic embryos for long-term germplasm storage

Cryopreservation using encapsulation-dehydration was developed for the long-term conservation of cocoa (Theobroma cacao L.) germplasm. Survival of individually encapsulated somatic embryos after desiccation and cryopreservation was achieved through optimization of cryoprotectants (abscisic acid (ABA) and sugar), duration of osmotic and evaporative dehydration, and embryo development stage. Up to 63% of the genotype SPA4 early-cotyledonary somatic embryos survived cryopreservation following 7 days preculture with 1 M sucrose and 4 h silica exposure (16% moisture content in bead). This optimized protocol was successfully applied to three other genotypes, e.g. EET272, IMC14 and AMAZ12, with recovery frequencies of 25, 40 and 72%, respectively (but the latter two genotypes using 0.75 M sucrose). Recovered SPA4 somatic embryos converted to plants at a rate of 33% and the regenerated plants were phenotypically comparable to non-cryopreserved somatic embryo-derived plants.

Photocatalytic degradation of humic acid in saline waters. Part 1. Artificial seawater: influence of TiO2, temperature, pH, and air-flow

We report the first systematic study on the photocatalytic oxidation of humic acid (HA) in artificial seawater (ASW). TiO2 (Degussa P25) dispersions were used as the catalyst with irradiation from a medium-pressure mercury lamp. The optimum quantity of catalyst was found to be between 2 and 2.5 g l(-1); whiled the decomposition was fastest at low pH values (pH 4.5 in the range examined), and the optimum air-flow, using an immersion well reactor with a capacity of 400 ml, was 850 ml min(-1). Reactivity increased with air-flow up to this figure, above which foaming prevented operation of the reactor. Using pure. oxygen, an optimal flow rate was observed at 300 nil min(-1), above which reactivity remains essentially constant. Following treatment for 1 h, low-salinity water (2700 mg l(-1)) was completely mineralised, whereas ASW (46000 mg l(-1)) had traces of HA remaining. These effects are interpreted and kinetic data presented. To avoid problems of precipitation due to change of ionic strength humic substances were prepared directly in ASW, and the effects of ASW on catalyst suspension and precipitation have been taken into account. The Langmuir-Hinshelwood kinetic model has been shown to be followed only approximately for the catalytic oxidation of HA in ASW. The activation energy for the reaction derived from an Arrhenius treatment was 17 ( +/-0.6) kJ mol(-1). (C) 2003 Elsevier Science Ltd. All rights reserved.

An in situ time-dependent study of the photodimerisation of chloro-derivatives of trans-cinnamic acid using infrared microspectroscopy with a synchrotron radiation source

The photodimerisation of single crystals of substituted cinnamic acid has been monitored continuously by infrared microscopy using a synchrotron source. The beta-form of 2,4-dichloro-trans-cinnamic acid dimerises under ultraviolet irradiation to form the corresponding beta-truxinic acid derivative in a reaction which follows strictly first order kinetics. By contrast the corresponding reactions in single crystals of beta-2-chloro-trans-cinnamic acid and beta-4-chloro-trans-cinnamic acid deviate somewhat from first order kinetics as a result of solid-state effects. In all three cases the reactions proceed smoothly from monomer to dimer with no hint of any reaction intermediate.

The photodimerisation of the alpha- and beta-forms of trans-cinnamic acid: a study of single crystals by vibrational microspectroscopy

Infrared and Raman microspectroscopy have been used to follow the photodimerisation reactions of single crystals, the alpha- and beta-forms of trans-cinnamic acid. This approach allows the starting materials and products -alpha-truxillic acid that has C-i symmetry and beta-truxinic acid, which has C-s symmetry-to be identified. It also allows the topotactic nature of the reaction to be confirmed. Attempts to produce the poorly-defined unreactive gamma-form of trans-cinnamic acid resulted only in a mixture of the alpha- and beta-forms. The findings suggest a wide role for these spectroscopic methods in monitoring solid-state organic reactions. (C) 2002 Elsevier Science B.V. All rights reserved.

Structural and equilibrium studies on mixed ligand complexes of Co(II), Ni(II), Cu(II) and Zn(II) with N-(2-benzimidazolyl)methyliminodiacetic acid and typical N,N donor ligands

Mixed ligand complexes: [Co(L)(bipy)] (.) 3H(2)O (1), [Ni(L)(phen)] (.) H2O (2), [Cu(L)(phen)] (.) 3H(2)O (3) and [Zn(L)(bipy)] (.) 3H(2)O (4), where L2- = two -COOH deprotonated dianion of N-(2-benzimidazolyl)methyliminodiacetic acid (H(2)bzimida, hereafter, H,L), bipy = 2,2' bipyridine and phen = 1,10-phenanthroline have been isolated and characterized by elemental analysis, spectral and magnetic measurements and thermal studies. Single crystal X-ray diffraction studies show octahedral geometry for 1, 2 and 4 and square pyramidal geometry for 3. Equilibrium studies in aqueous solution (ionic strength I = 10(-1) mol dm(-3) (NaNO3), at 25 +/- 1 degrees C) using different molar proportions of M(II):H2L:B, where M = Co, Ni, Cu and Zn and B = phen, bipy and en (ethylene diamine), however, provides evidence of formation of mononuclear and binuclear binary and mixed ligand complexes: M(L), M(H-1L)(-), M(B)(2+), M(L)(B), M(H-1L)(B)(-), M-2(H-1L)(OH), (B)M(H-1L)M(B)(+), where H-1L3- represents two -COOH and the benzimidazole NI-H deprotonated quadridentate (O-, N, O-, N), or, quinquedentate (O-, N, O-, N, N-) function of the coordinated ligand H,L. Binuclear mixed ligand complex formation equilibria: M(L)(B) + M(B)(2+) = (B)M(H-1L)M(B)(+) + H+ is favoured with higher pi-acidity of the B ligands. For Co(II), Ni(II) and Cu(II), these equilibria are accompanied by blue shift of the electronic absorption maxima of M(II) ions, as a negatively charged bridging benzimidazolate moiety provides stronger ligand field than a neutral one. Solution stability of the mixed ligand complexes are in the expected order: Co(II) < Ni(II) < Cu(II) > Zn(II). The Delta logK(M) values are less negetive than their statistical values, indicating favoured formation of the mixed ligand complexes over the binary ones. (c) 2005 Elsevier B.V. All rights reserved.

Difluorinated analogues of shikimic acid

Investigations into the quinate to shikimate transformation have been carried out, the results of which have been exploited in the synthesis of a novel difluoromethylene homologue of shikimic acid from (-)-quinic acid. Martin's sulfurane {Ph2S[OC(CF3)(2)Ph](2)} was the reagent of choice for the key dehydration step of this synthesis. The results of investigations into the synthesis of the important natural product analogue, 6,6-difluoroshikimic acid are also reported. (C) 2003 Elsevier Science Ltd. All rights reserved.

Synthesis and characterization of hyperbranched polyesters incorporating the AB(2) monomer 3,5-bis(3-hydroxylprop-1-ynyl)benzoic acid

The AB, monomer, 3,5-bis(3-hydroxylprop-1-ynyl)benzoic acid 1, has been synthesized using a Sonogashira cross-coupling with a palladium catalyst system developed for use with deactivated aryl halides. Numerous condensation methods have then been assessed in the homopolymerization of the acid-diol monomer 1 to afford hyperbranched polyesters. However, as a result of the thermal instability of the monomer, direct thermal polymerizations could not be employed. Alternative approaches using carbodiimide-coupling reagents enabled the production of soluble polyesters possessing molecular weights and degrees of branching ranging from 2500 to 11,000 and 0.22 to 0.33, respectively. (C) 2003 Elsevier Ltd. All rights reserved.

In vivo transformations of dihydroartemisinic acid in Artemisia annua plants

[15-(CH3)-C-13-H-2]-dihydroartemisinic acid (2a) and [15-(CH3)-H-2]-dihydroartemisinic acid (2b) have been fed via the root to intact Artemisia annua plants and their transformations studied in vivo by one-dimensional H-2 NMR spectroscopy and two-dimensional, C-13-H-2 correlation NMR spectroscopy (C-13-(2) H COSY). Labelled dihydroartemisinic acid was transformed into 16 12-carboxy-amorphane and cadinane sesquiterpenes within a few days in the aerial parts of A. annua, although transformations in the root were much slower and more limited. Fifteen of these 16 metabolites have been reported previously as natural products from A. annua. Evidence is presented that the first step in the transformation of dihydroartemisinic acid in vivo is the formation of allylic hydroperoxides by the reaction of molecular oxygen with the Delta(4,5)-double bond in this compound. The origin of all 16 secondary metabolites might then be explained by the known further reactions of such hydroperoxides. The qualitative pattern for the transformations of dihydroartemisinic acid in vivo was essentially unaltered when a comparison was made between plants, which had been kept alive and plants which were allowed to die after feeding of the labelled precursor. This, coupled with the observation that the pattern of transformations of 2 in vivo demonstrated very close parallels with the spontaneous autoxidation chemistry for 2, which we have recently demonstrated in vitro, has lead us to conclude that the main 'metabolic route' for dihydroartemisinic acid in A. annua involves its spontaneous autoxidation and the subsequent spontaneous reactions of allylic hydroperoxides which are derived from 2. There may be no need to invoke the participation of enzymes in any of the later biogenetic steps leading to all 16 of the labelled 11,13-dihydro-amorphane sesquiterpenes which are found in A. annua as natural products. (C) 2003 Elsevier Ltd. All rights reserved.

Synthesis of labelled dihydroartemisinic acid

[15-(CH3)-C-13-H-2]-Dihydroartemisinic acid (2a), [15-(CH3)-H-2]-dihydroartemisinic acid (2b) and [15-(CH3)-C-13]-dihydroartemisinic acid (2c) have been obtained in good yield and high isotopic enrichment by a reconstructive synthesis from artemisinin. These labelled compounds were designed to be used in biosynthetic experiments to determine the origins of artemisinin and other sesquiterpene natural products from Artemisia annua. (C) 2003 Elsevier Ltd. All rights reserved.

In vivo transformations of artemisinic acid in Artemisia annua plants

Artemisinic acid labeled with both C-13 and H-2 at the 15-position has been fed to intact plants of Artemisia annua via the cut stem, and its in vivo transformations studied by 1D- and 2D-NMR spectroscopy. Seven labeled metabolites have been isolated, all of which are known as natural products from this species. The transformations of artemisinic acid-as observed both for a group of plants, which was kept alive by hydroponic administration of water and for a group, which was allowed to die by desiccation-closely paralleled those, which have been recently described for its 11,13-dihydro analog, dihydroartemisinic acid. It seems likely therefore that similar mechanisms, involving spontaneous autoxidation of the Delta(4,5) double bond in both artemisinic acid and dihydroartemisinic acid and subsequent rearrangements of the resultant allylic hydroperoxides, may be involved in the biological transformations, which are undergone by both compounds. All of the sesquiterpene metabolites, which were obtained from in vivo transformations of artemisinic acid retained their unsaturation at the 11,13-position, and there was no evidence for conversion into any 11,13-dihydro metabolite, including artemisinin, the antimalarial drug, which is produced by A. annua. This observation led to the proposal of a unified biosynthetic scheme, which accounts for the biogenesis of many of the amorphane and cadinane sesquiterpenes that have been isolated as natural products from A. annua. In this scheme, there is a bifurcation in the biosynthetic pathway starting from amorpha-4,11-diene leading to either artemisinic acid or dihydroartemisinic acid; these two committed precursors are then, respectively, the parents for the two large families of highly oxygenated 11,13-dehydro and 11,13-dihydro sesquiterpene metabolites, which are known from this species. (C) 2007 Elsevier Ltd. All rights reserved.