Binding of polyphenols to plant cell wall analogues - Part 1: Anthocyanins

Anthocyanins are located within the vacuole of plant cells, and are released following cell rupture during eating or processing at which time they first come into contact with the plant cell wall. The extent of anthocyanin-cell wall interaction was investigated by monitoring the rate of anthocyanin depletion in the presence of pure cellulose or cellulose-pectin composites as cell wall models. It was found that anthocyanins interact with both cellulose and pectin over a two-stage process with initially (mins-hours) 13 similar to 18% of anthocyanins binding to cellulose or cellulose/pectincomposites. With prolonged exposure (days-weeks), a gradual increase in anthocyanin binding occurs, possibly due to anthocyanins stacking on top of a base layer. Binding of acylated and non-acylated anthocyanins followed a similar pattern with slightly more (5-10%) binding of the acylated forms. Composites with the highest pectin content had the greatest anthocyanin binding suggesting the existence of both ionic interactions (with pectin) and hydrophobic interactions (with cellulose) of anthocyanin with plant cell walls.

Pro-apoptotic NOXA is implicated in atmospheric-pressure plasma-induced melanoma cell death

Atmospheric-pressure plasma (APP) has been successfully used to treat several types of cancers in vivo and in vitro, with the effect being primarily attributed to the generation of reactive oxygen species (ROS). However, the mechanisms by which APP induces apoptosis in cancer cells require further elucidation. In this study, the effects of APP on the expression of 500 genes in melanoma Mel007 cancer cells were examined. Pro-apoptotic phorbol-12-myristate-13-acetate-induced protein (PMAIP1), also known as NOXA, was highly expressed as a result of APP treatment in a dose-dependent manner. Blocking of ROS using scavenger NAC or silencing of NOXA gene by RNA interference inhibited the APP-induced NOXA genes upregulation and impaired caspases 3/7 mediated apoptosis, confirming the important role plasma-generated ROS species and pro-apoptotic NOXA play in APP-induced cancer cell death.

A new magnesium-air cell for long-life applications

A novel type of magnesium-air primary cell has been evolved which employs non-polluting and abundantly available materials. The cell is based on the scheme Mg/Mg(NO3)2, NaNO2, H20/Q(C). The magnesium anode utilization is about 90% at a current density of 20 mAcm -2. The anode has been shown to exhibit a low open-circuit corrosion, a relatively uniform pattern of corrosion and a low negative difference effect in the electrolyte developed above as compared to the conventional halide or perchlorate electrolytes. In the usual air-depolarized mode of operation, the cell has been found to be capable of continuous discharge over several months at a constant cell voltage of about 1 V and a current density of 1 mAcm -2 at the cathode. The long service-life capability arises from the formation of a protective film on the porous carbon cathode and fast sedimentation of the anodic product (magnesium hydroxide) in the electrolyte. The cell has a shelf-life in the activated state of about a year due to the low open-circuit corrosion of the anode. These favourable features suggest the practical feasibility of developing economical, long-life, non-reserve magnesium-air ceils for diverse applications using magnesium anodes with a high surface area and porous carbon-air electrodes.

Lack of release of bound anthocyanins and phenolic acids from carrot plant cell walls and model composites during simulated gastric and small intestinal digestion

Separately, polyphenols and plant cell walls (PCW) are important contributors to the health benefits associated with fruits and vegetables. However, interactions with PCW which occur either during food preparation or mastication may affect bioaccessibility and hence bioavailability of polyphenols. Binding interactions between anthocyanins, phenolic acids (PAs) and PCW components, were evaluated using both a bacterial cellulose-pectin model system and a black carrot puree system. The majority of available polyphenols bound to PCW material with 60-70% of available anthocyanins and PAs respectively binding to black carrot puree PCW matter. Once bound, release of polyphenols using acidified methanol is low with only similar to 20% of total anthocyanins to similar to 30% of PAs being released. Less than 2% of bound polyphenol was released after in vitro gastric and small intestinal (S.I.) digestion for both the model system and the black carrot puree PCW matter. Confocal laser scanning microscopy shows localised binding of anthocyanins to PCW. Very similar patterns of binding for anthocyanins and PAs suggest that PAs form complexes with anthocyanins and polysaccharides. Time dependent changes in extractability with acidified methanol but not the total bound fraction suggests that initial nonspecific deposition on cellulose surfaces is followed by rearrangement of the bound molecules. Minimal release of anthocyanins and PAs after simulated gastric and S.I. digestion indicates that polyphenols in fruits and vegetables which bind to the PCW will be transported to the colon where they would be expected to be released by the action of cell wall degrading bacteria.

Binding of polyphenols to plant cell wall analogues - Part 2: Phenolic acids

Bacterial cellulose and cellulose-pectin composites were used as well-defined model plant cell wall (PCW) systems to study the interaction between phenolic acids (PA) derived from purple carrot juice concentrate (PCJC) and PCW components. Significant PA depletion from solution occurred, with pure cellulose initially (30 s-1 h) absorbing more than cellulose-pectin composites in the first hour (ca 20% cf 10-15%), but with all composites absorbing similar levels (ca 30%) after several days. Individual PAs bound to different relative extents with caffeic acid > chlorogenic acid > ferulic acid. Extrapolation of data for these model systems to carrot puree suggests that nutritionally-significant amounts of PAs could bind to cell walls, potentially restricting bioavailability in the small intestine and, as a consequence, delivering PAs to the large intestine for fermentation and metabolism by gut bacteria. (C) 2012 Elsevier Ltd. All rights reserved.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Stunted cotton (Gossypium hirsutum L.) fully recovers biomass and yield of seed cotton after delayed root inoculation with spores of an arbuscular mycorrhizal fungus (Glomus mosseae)

In the northern grain and cotton region of Australia, poor crop growth after long periods of fallow, called 'long-fallow' disorder, is caused by a decline of natural arbuscular-mycorrhizal fungi (AMF). When cotton was grown in large pots containing 22 kg of Vertisol from a field recently harvested from cotton in Central Queensland, plants in pasteurised soil were extremely stunted compared with plants in unpasteurised soil. We tested the hypothesis that this extreme stunting was caused by the absence of AMF and examined whether such stunted plants could recover from subsequent treatment with AMF spores and/or P fertiliser. At 42 days after sowing, the healthy cotton growing in unpasteurised soil had 48% of its root-length colonised with AMF, whereas the stunted cotton had none. After inoculation with AMF spores (6 spores/g soil of Glomus mosseae) and/or application of P fertiliser (50 mg P/kg soil) at 45 days after sowing, the stunted plants commenced to improve about 25 days after treatment, and continued until their total dry matter and seed cotton production equalled that of plants growing in unpasteurised soil with natural AMF. In contrast, non-mycorrhizal cotton grown without P fertiliser remained stunted throughout and produced no bolls and only 1% of the biomass of mycorrhizal cotton. Even with the addition of P fertiliser, non-mycorrhizal cotton produced only 64% of the biomass and 58% of the seed cotton (lint + seed) of mycorrhizal cotton plants. These results show that cotton is highly dependent on AMF for P nutrition and growth in Vertisol (even with high rates of P fertiliser), but can recover from complete lack of AMF and consequent stunting during at least the first 45 days of growth when treated with AMF spores and/or P fertiliser. This corroborates field observations in the northern region that cotton may recover from long-fallow disorder caused by low initial levels of AMF propagules in the soil as the AMF colonisation of its roots increases during the growing season.

Evidence for Non-coordinated synthesis of 5 S RNA in the thermophilic fungus, Thermomyces lanuginosus

Analysis of ribosomes and the post ribosomal supernatant fraction of actively growing cells ofThermomyces lanuginosus showed the presence of free 5 S RNA in the supernatant fraction. This 5 S RNA was identical to the ribosomal 5 S RNA in its electrophoretic mobility on 10% Polyacrylamide gel and in its base composition. 5 S RNA from both the sources gave evidence for the presence of diphosphate at the 5’ end. Most of the 5 S RNA that appeared in the cytoplasm was that transported from the nucleus during the isolation. This could be prevented by the use of a hexylene glycol-HEPES buffer.

Biological control of Mikania micrantha in Papua New Guinea and Fiji using the rust fungus Puccinia spegazzinii

Mikania micrantha Kunth (Asteraceae), commonly known as ‘mile-a-minute’, is a neotropical plant species now found in 17 Pacific island countries and territories, invading small cropping areas and plantations, thereby reducing productivity and food security. In 2006, a biocontrol project on M. micrantha commenced in Fiji and Papua New Guinea (PNG). The distribution of M. micrantha as well as baseline data such as plant growth rates and socio-economic impacts were determined before the importation of any biocontrol agents. Mikania micrantha was recorded in all 15 lowland provinces in PNG and on all major islands in Fiji. Plants grow about 3.2cm/day in PNG and about 1.9cm/day in Fiji. A socio-economic survey, involving over 370 respondents in over 220 villages from 15 provinces in PNG, found that 78% of respondents considered M. micrantha a serious weed and about 44% had M. micrantha, which they needed to weed at least fortnightly, in over a third of their land. Over 80% of respondents used slashing and/or handpulling as the preferred method of weed control. About 40% of respondents considered that M. micrantha reduced crop yield by more than 30%. In Fiji, 52 respondents from four islands participated in the survey. Over 60% of respondents in Fiji considered M. micrantha a serious weed and 23% had about 30% of their farm lands infested with the weed. Only 15% of respondents needed to weed at least fortnightly, with 56% using slashing and/or hand-pulling as the preferred means of control. Over 65% of respondents estimated that they lost at least 30% of potential crop yield to M. micrantha. Nearly 90% of respondents used M. micrantha as a medicinal plant to treat cuts and wounds. The life history of the rust Puccinia spegazzinii de Toni (Pucciniales: Pucciniaceae), originating from Ecuador, and imported into PNG and Fiji in 2008, was studied. P. spegazzinii is a microcyclic and autoecious rust and has a life cycle of 18-22 days. An efficient culturing and field release method was developed. Since 2008, the rust has been released at over 450 sites in 15 provinces in PNG, establishing at nearly 70 sites in four provinces. From some sites, the rust has spread over 7 km in 12 months. In Fiji, the rust has been released at over 80 sites, on four of the main islands, namely Viti Levu, Vanua Levu, Taveuni and Ovalau, and has established at 20 sites on Viti Levu and Vanua Levu. Plant growth studies and field monitoring in PNG showed that P. spegazzinii can significantly reduce the growth and density of M. micrantha and offers great potential for the control of this weed.

Isolation and Culture of a Thermophilic Fungus, Melanocarpus albomyces, and Factors Influencing the Production and Activity of Xylanase

Summary: An uncommon thermophilic fungus, Melanocarpus albomyces, was isolated from soil and compost by incubating samples in a glucose/sorbose/asparagine liquid medium, followed by enrichment culture in medium containing sugarcane bagasse as carbon source. The culture filtrate protein of the fungus grown in the presence of bagasse or xylose hydrolysed xylan and some other polysaccharides but cellulose was not hydrolysed. High extracellular xylanase (EC 3.2.1.8) activity was produced by cultures grown on xylose or hemicellulosic materials. The enzyme was induced in glucose-grown washed mycelia in response to addition of xylose or xylan but not by alkyl or aryl β-D-xylosides. Cultures produced higher enzyme yields in shaken flasks than in a fermenter. Gel-filtration chromatography of culture filtrate protein showed the presence of two isoenzymes of xylanase, whose relative proportions varied with the carbon source used for growth. The extent of hydrolysis of heteroxylans or the hemicellulosic fraction of bagasse by culture filtrate protein preparations was greater when the cultures had been grown on bagasse rather than xylose as the inducing substrate. The activity of xylanase preparations was increased when an exogenous β-glucosidase was added.

Assessing the potential of the rust fungus Puccinia spegazzinii as a classical biological control agent for the invasive weed Mikania micrantha in Papua New Guinea

The rust fungus Puccinia spegazzinii was introduced into Papua New Guinea (PNG) in 2008 as a classical biological control agent of the invasive weed Mikania micrantha (Asteraceae), following its earlier release in India, mainland China and Taiwan. Prior to implementing field releases in PNG, assessments were conducted to determine the most suitable rust pathotype for the country, potential for damage to non-target species, most efficient culturing method and potential impact to M. micrantha. The pathotype from eastern Ecuador was selected from the seven pathotypes tested, since all the plant populations evaluated from PNG were highly susceptible to it. None of the 11 plant species (representing eight families) tested to confirm host specificity showed symptoms of infection, supporting previous host range determination. A method of mass-producing inoculum of the rust fungus, using a simple technology which can be readily replicated in other countries, was developed. Comparative growth trials over one rust generation showed that M. micrantha plants infected with the rust generally had both lower growth rates and lower final dry weights, and produced fewer nodes than uninfected plants. There were significant correlations between the number of pustules and (a) the growth rate, (b) number of new nodes and (c) final total dry weight of single-stemmed plants placed in open sunlight and between the number of pustules and number of new nodes of multi-stemmed plants placed under cocoa trees. The trials suggest that field densities of M. micrantha could be reduced if the rust populations are sufficiently high. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved.

Monitoring of Fungal Growth and Degradation of Wood

Valko- ja ruskolahosienet tunnetaan luonnossa tehokkaimpina puun ja karikkeen lignoselluloosan lahottajina. Valkolahosienet pystyvät hajottamaan kaikkia puun osia: ligniiniä, selluloosaa ja hemiselluloosaa. Selektiivisesti ligniiniä hajottavat sienet lahottavat puusta suhteessa enemmän vaikeasti hajoavaa ligniiniä kuin selluloosaa tai hemiselluloosaa, jolloin jäljelle jää valkoista ja miltei puhdasta selluloosaa. Bioteknisissä sovelluksissa juuri selektiviiviset valkolahottajat ovat kiinnostavia. Niiden avulla voidaan puuhaketta esikäsitellä esimerkiksi paperinvalmistuksessa haitallisen ligniinin poistamiseksi. Ruskolahosienet ovat huomattavia puun, puutavaran ja puisten rakenteiden lahottajia, kuten tässä työssä käytetty Gloeophyllum trabeum (saunasieni ) ja Poria (Postia) placenta (istukkakääpä). Ruskolahosienet hajottavat puusta hemiselluloosan lisäksi selluloosaa, jolloin jää jäljelle ruskea ja jauhomaiseksi mureneva ligniini. Ruskolahosienet muovaavat ligniiniä jonkin verran. Kahden ruskolahosienen G. trabeumin ja P. placentan lisäksi tutkittiin valkolahosieniä, joista Ceriporiopsis subvermispora (karstakääpä) ja harvinainen Physisporinus rivulosus -sieni (talikääpä) hajottavat ligniiniä erittäin selektiivisesti. Phanerochaete chrysosporium on kaikkialla paljon tutkittu sieni, ja Phlebia radiata valkolahosientä (rusorypykkä) on tutkittu paljon mikrobiologian osastolla. Lisäksi tutkittiin Phlebia tremellosa -sienten (hytyrypykkä) ligninolyyttisten entsyymien tuottoa ja 14C-leimatun synteettisen ligniinin (DHP) hajotusta. P. radiata ja P. tremellosa -sienten on todettu aiemmin hajottavan ligniiniä selektiivisesti. Työssä selvitettiin miten sienten kasvua voi mitata, miten vertailukelpoisia eri mittaamismenetelmillä saadut tulokset ovat ja ilmenevätkö sienten aktiivisimmat kasvuvaiheet samaan aikaan eri menetelmillä mitattuna. Tärkeimmät tulokset olivat seuraavat havainnot: (i) P. radiata ja P. tremellosa -sienikannat tuottivat ligniini- ja mangaaniperoksidaasientsyymejä (LiP ja MnP) sekä lakkaasia, ja sienistä puhdistettiin 2-3 LiP- ja P. radiatasta yksi MnP-entsyymi; (ii) P. tremellosa -sienet hajottivat leimattua synteettistä ligniiniä (DHP) yhtä hyvin kuin paljon tutkitut P. chrysosporium ja P. radiata -sienet; (iii) puu, sienen luonnollinen kasvualusta, lisäsi valkolaho- ja ruskolahosienten demetoksylaatiota [O14CH3]-leimatusta ligniinin malliyhdisteestä 14CO2:ksi ilman puuta olleeseen alustaan verrattuna; (iv) demetoksylaatio (14CO2:n tuotto) oli normaalissa ilma-atmosfäärissä useimmiten parempi happeen verrattuna; (v) hapessa paras 14CO2:n tuotto saatiin puupalakasvatuksissa, joihin oli lisätty ravinnetyppeä tai typen lisäksi glukoosia sekä valkolaho- että ruskolahosienillä; (vi) ilmassa 14CO2:n tuotto oli puulla voimakkainta valkolahosienillä ilman lisäravinteita, kun taas G. trabeum -sienellä se oli yhtä hyvä eri alustoissa; (vii) biomassan muodostuminen rihmastojen ergosterolipitoisuuksista mitattuna oli ruskolahosienillä parempi kuin valkolahosienillä; (viii) ja biomassojen huippupitoisuudet olivat 6:lla sienellä eri suuruisia ja niiden maksimimäärien ajankohdat vaihtelivat viiden viikon kasvatusten kuluessa. Mikrobiologian osastolla Viikissä eristetty ja paljon tutkittu P. radiata -valkolahosieni oli mukana kaikissa tehdyissä kokeissa. Sienen LiP-aktiivisuus ja 14CO2:n tuotto 14C-rengas-leimatusta synteettisestä ligniinistä (DHP) korreloivat erittäin hyvin. Biomassan muodostuminen ergosterolilla määritettynä tuki hyvin entsyymiaktiivisuusmittauksilla ja isotooppikasvatuksilla saatuja tuloksia.