Growth and nutrient uptake of tea under different aluminium concentrations

BACKGROUND: The uptake of essential nutrients such as P, Fe, K, Ca and Mg is depressed by Al in most plants. This study aimed to investigate the concentrations at which Al could be toxic to C. sinensis. The suppression of nutrient uptake was investigated by comparing growth and nutrient uptake at different Al doses. The quantification of Al in apoplasm, symplasm and cell wall of C. sinensis was also studied. RESULTS: In the absence of Al, the growth of C. sinensis was retarded. Test doses over 1 mM Al were toxic to C. sinensis. At concentrations of 0.25 or 0.5 mM, distinct rhizostimulation was noted, and within a short period (2 weeks), the biomass of these seedlings increased by 44 and 35%, respectively, compared to 0 and 14% in control and 1 mM Al, respectively. In general, at beneficial doses (0.25, 0.5), Al stimulated the uptake of Ca, Mg, K and Mn, whereas the uptake of Fe, Cu and Zn was retarded. Fine roots of the seedlings had the highest levels of Al, compared to leaves, branches and main roots. In the root tips, most of the Al was present in the soluble fractions of the apoplasm and symplasm, and very low levels of Al was bound to the cell walls, which was in good agreement with the observed mobility of Al in C. sinensis. CONCLUSION: The results of the present study support the view that Al plays a nutritive role for C. sinensis. The rhizostimulatory effects of Al on C. sinensis have been explained as a consequence of enhanced nutrient uptake. Copyright © 2008 Society of Chemical Industry     

Impact of lignin composition on cell‐wall degradability in an Arabidopsis mutant

An Arabidopsis mutant that does not deposit syringyl‐type lignin was used to test the hypothesis that lignin composition impacts cell‐wall degradability. Two lines of the ferulate‐5‐hydroxylase‐deficient fah1 mutant and the wild‐type control line were grown in the greenhouse. In Experiment 1, the plants were harvested at the mature seed stage. For Experiment 2, plants were harvested 5, 6, 7 and 8 weeks after sowing. In both experiments stems were collected and analysed for cell‐wall concentration and composition, and in vitro degradability of cell‐wall polysaccharide components by rumen micro‐organisms. The absence of syringyl‐type lignin was confirmed for the mutant lines by nitrobenzene oxidation and pyrolysis‐GC‐MS. Lignin concentration was the same for all three Arabidopsis lines, at all stages of maturity. The Arabidopsis stems were similar to forage legumes in that the potentially degradable cell‐wall fraction was very quickly degraded. Cell‐wall polysaccharide degradability did not differ among the Arabidopsis lines in the first experiment after 24‐h fermentations, but the cell‐wall polysaccharides of the fah1‐2 mutant line were less degradable after 96‐h than either the wild‐type or the fah1‐5 mutant. In contrast, in Experiment 2 no differences among lines were found for cell‐wall polysaccharide degradability after either 24‐ or 96‐h fermentations; however, signficantly higher levels of ester‐bound ferulic acid were found in the walls of the fah1 mutant lines. As expected, increasing stem maturity was correlated with reduced degradation of cell‐wall polysaccharides. These experiments indicate that either lignin composition, as measured by syringyl‐to‐guaiacyl ratio, does not alter cell‐wall degradability in Arabidopsis, or that the fah1 mutation has other effects on the cell walls of these mutants such that the impact of the change in syringyl‐to‐guaiacyl ratio is masked. © 1999 Society of Chemical Industry     

Development of an Effective Treatment for A 5‐Log Reduction of Escherichia coli in Refrigerated Pickle Products

Refrigerated cucumber pickle products cannot be heat processed due to the loss of characteristic sensory attributes. Typically brined refrigerated pickles contain less than 100 mM acetic acid with pH values of 3.7 to 4.0. Refrigeration (4 to 10 °C) helps to inhibit the growth of spoilage bacteria and maintain flavor, texture, and appearance of the pickles. Previous research has shown that pathogenic Escherichia coli strains are unusually acid resistant and survive better in refrigerated acid solutions than at higher temperatures. We found that E. coli O157:H7 can survive for 1 mo or longer at 4 °C in brines typical of commercial refrigerated pickles. Our objective was to develop methods to assure a 5‐log reduction of pathogenic E. coli in these types of products, while maintaining the sensory characteristics. A novel brine formulation was developed, based on current commercial refrigerated pickle brines, which contained 25 mM fumaric acid, 5 mM benzoic acid, 70 mM acetic acid, and 342 mM (2%) sodium chloride, with a pH of 3.8. Sensory data indicate that this formulation did not affect flavor or other sensory attributes of the product, compared to traditional formulations. We achieved a 5‐log reduction of E. coli O157:H7 at 30 °C for 1.52 ± 0.15 d, at 20 °C for 3.12 ± 0.34 d, or at 10 °C for 8.83 ± 0.56 d. Growth of lactic acid bacteria was also inhibited. These results can be used by manufacturers to assure a 5‐log reduction in cell numbers of E. coli O157:H7 and Salmonella without a heat process during the manufacture of refrigerated pickle products. Practical Application : While refrigerated acidified vegetable products are exempt from the acidified foods regulations, we have shown that the vegetative microbial pathogens E. coli O157:H7 can survive for up to 1 mo in these products, given current commercial production practices. To improve the safety of refrigerated pickle products, a brine formulation with reduced acetic acid, but containing fumaric acid, was developed to assure a 5‐log reduction in cell numbers of E. coli O157:H7 without a heat process. The formulation can be used to assure the safety of refrigerated pickled vegetables without altering sensory characteristics.     

Curtailing Oxidation‐Induced Loss of Myosin Gelling Potential by Pyrophosphate Through Shielding the S1 Subfragment

In muscle food processing, where oxidation is inevitable, phosphates are usually added to improve water binding. This present study attempted to investigate the interactive roles of protein oxidation and pyrophosphate (PP) during thermal gelation of myosin. Myosin isolated from pork muscle was solubilized in 0.5 M NaCl at pH 6.2 then oxidatively stressed with an iron‐redox cycling system that produces hydroxyl radicals with or without 1 mM PP and 2 mM MgCl₂ at 4 °C for 12 or 24 h then heated to 50 °C at 1.3 °C/min. Protein conformational stability was measured by differential scanning calorimetry, and covalent cross‐linking was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis following chymotrypsin digestion. The binding of PP to myosin suppressed disulfide bond formation in myosin subfragments 1 and 2 and partially inhibited oxidation‐initiated cross‐linking of heavy meromyosin during myosin gelation with a lesser effect on light meromyosin. In the presence of PP, myosin exhibited less loss of conformational integrity upon oxidation than myosin without PP. Rheological analysis from 20 to 75 °C indicated up to 32% decreases (P < 0.05) in elastic modulus (G′) of myosin gels due to oxidation. However, the presence of 1 mM PP, which also lowered the gelling capacity of myosin, inhibited the oxidation‐induced G′ by nearly half (P < 0.05). These results suggest that the protection of myosin head from oxidative modification by PP can be a significant factor for the minimization of gelling property losses during cooking of comminuted meats.     

Pb2+Inhibits Competitively Flocculation of Saccharomyces cerevisiae

Pb²⁺inhibited calcium‐induced flocculation in Flo1 (S646–1B) and NewFlo phenotype strains (NCYC 1190, NCYC 1195 and NCYC 1364) of Saccharomyces cerevisiae. Flocculation was restored after washing with water or EDTA, which suggests a reversible binding of Pb²⁺to yeast cell walls. Pb²⁺probably inhibited flocculation by competing with Ca²⁺, since Pb²⁺inhibition was alleviated by excess of Ca²⁺. Using a fluorescent avidinfluorescein isothiocyanate (Avidin‐FITC) probe, active cell surface flocculation lectins, in the presence of Ca²⁺ions, were visualized. Conversely, Avidin‐FITC was not fixed to yeast walls of flocculent cells, in the presence of Pb²⁺ions or in the simultaneous presence of 0.05 mM Ca²⁺and 0.4 mM Pb²⁺. These results suggest that Pb²⁺ions were not able to induce the correct conformation of the lectin‐like component and reinforces the hypothesis that Pb²⁺ions compete for the same “calcium site” of flocculation zymolectins.     

Effect of industrial processing on alfalfa cell walls

Thermal treatment is one of the widely used methods for feedstuff preservation. The aim of this study was to evaluate possible cell wall alterations during the industrial‐scale drying of alfalfa. Alfalfa is mainly dehydrated and pelleted using two drying treatments. First a thermal treatment at 70 °C is applied to the chopped raw material, prior to the main dehydration procedure which involves a temperature of about 600 °C for a restricted time (15 min). The different steps of the dehydration process did not cause significant changes in alfalfa cell wall composition, but the proportion of labile ether‐linked lignin structure (β‐O‐4) increased after the thermal shock. Chemical fractionation of cell wall carbohydrates showed that pectins were less accessible to extraction with ammonium oxalate after alfalfa drying. Moreover, xylanase (endo enzyme) was more effective on cell walls obtained from heated material. These results suggested that heat processing causes some changes in interpolymer bonds (covalent and hydrogen bonds) between pectins and the lignin/hemicellulose matrix, whereby the cell wall network is modified. © 2002 Society of Chemical Industry     

Dual functions of Mdt1 in genome maintenance and cell integrity pathways in Saccharomyces cerevisiae

Recent evidence indicates considerable cross‐talk between genome maintenance and cell integrity control pathways. The RNA recognition motif (RRM)‐ and SQ/TQ cluster domain (SCD)‐containing protein Mdt1 is required for repair of 3′‐blocked DNA double‐strand breaks (DSBs) and efficient recombinational maintenance of telomeres in budding yeast. Here we show that deletion of MDT1 (PIN4/YBL051C) leads to severe synthetic sickness in the absence of the genes for the central cell integrity MAP kinases Bck1 and Slt2/Mpk1. Consistent with a cell integrity function, mdt1Δ cells are hypersensitive to the cell wall toxin calcofluor white and the Bck1–Slt2 pathway activator caffeine. An RRM‐deficient mdt1‐RRM0 allele shares the severe bleomycin hypersensitivity, inefficient recombinational telomere maintenance and slt2 synthetic sickness phenotypes, but not the cell wall toxin hypersensitivity with mdt1Δ. However, the mdt1‐RRM(3A) allele, where only the RNA‐binding site is mutated, behaves similarly to the wild‐type, suggesting that the Mdt1 RRM functions as a protein–protein interaction rather than a nucleic acid‐binding module. Surprisingly, in a strain background where double mutants are sick but still viable, bck1Δmdt1Δ and slt2Δmdt1Δ mutants differ in some of their phenotypes, consistent with the emerging concept of flexible signal entry and exit points in the Bck1–Mkk1/2–Slt2 pathway. Overall, the results indicate that Mdt1 has partially separable functions in both cell wall and genome integrity pathways. Copyright © 2009 John Wiley & Sons, Ltd.     

The efficiency and comparison of novel techniques for cell wall disruption in astaxanthin extraction from Haematococcus pluvialis

The efficiency of various techniques pulsed electric field (PEF), ultrasound (US), high‐pressure microfluidisation (HPMF), hydrochloric acid (HCl) and ionic liquids (ILs) for cell wall disruption in astaxanthin extraction from Haematococcus pluvialis was compared. The results indicated that ILs, HCl and HPMF treatment were shown the most efficient for cell disruption with more than 80% astaxanthin recovery. While the cell wall integrity of H. pluvialis cyst cell was less affected by US and PEF treatment. It was found that imidazolium‐based ILs showed the greater potential for cell disruption than pyridinium‐based and ammonium‐based ILs. Among all the ILs examined, 1‐butyl‐3‐methylimidazolium chloride ([Bmim] Cl) exhibited efficient cell disruption and capability of astaxanthin recovery at mild condition (pretreatment with 40% IL aqueous solution at 40 °C, followed by extraction with methanol at 50 °C) without extensive energy consumption and special facility requirement. In addition, recyclability of ILs was excellent.     

Purification and characterisation of carrot (Daucus carota L) pectinesterase

A pectinesterase isoform with an alkaline isoelectric point of over 8.66 was detected in crude extracts of carrot. The enzyme was purified by ion exchange and molecular exclusion chromatography. The molecular weight of the isoform was 25 kDa, determined in native conditions by filtration through Sephadex G‐75 SF. The enzyme showed a high affinity for its substrate, with K_m and V_max values of 0.031 mg ml^?1 and 6.77 units respectively for apple pectin. The pectinesterase activity exhibited an optimum around pH 7.4 and was activated by metallic ions, with optimum activities at NaCl concentrations between 130 and 330 mM and at CaCl₂ concentrations between 15 and 50 mM . The enzyme was activated most by Ca²⁺ and exhibited a greater tolerance of high concentrations of Na⁺. Comparison of its heat stability with other pectinesterases of vegetable origin indicated that the purified isoform was very thermolabile, being rendered inactive by heating for 5 min at 70 °C. The enzyme was inhibited by high concentrations of polygalacturonic acid and competitively inhibited by D ‐galacturonic acid, with a K_i value of 1 mM . Copyright © 2003 Society of Chemical Industry     

Amino acids stimulate Akt phosphorylation,and reduce IL‐8 production and NF‐κB activity in HepG2 liver cells

Hepatic insulin resistance and inflammatory cytokine production contribute to the manifestation of the metabolic syndrome. As amino acids have been implicated in modulating insulin signaling and inflammation, we have investigated the effects of glutamine, leucine and proline on markers of inflammation and insulin sensitivity in HepG2 liver cells. Cells were incubated with IL‐1β (5 ng/mL) to stimulate IL‐8 production. After 24 h, glutamine inhibited IL‐8 production significantly (p<0.05) at 2, 5 and 10 mM (to 82, 73 and 72% of control), whereas leucine reduced IL‐8 production significantly only at 10 mM (66%) and proline at 5 and 10 mM (71 and 52%). Glutamine, leucine and proline all reduced NF‐κB activity after 3 h of IL‐1β stimulation at 2, 5 and 10 mM (p<0.001). Insulin‐induced (1 nM) Akt phosphorylation was reduced in cells treated with tumour necrosis factor‐α (10 ng/mL) for 24 h, but was partly restored by simultaneous incubation with glutamine, leucine and proline (25 mM). Phosphorylation of glycogen synthase kinase‐3β was unaffected by insulin stimulation and amino acid treatment. Our results indicate that glutamine, leucine and proline attenuate IL‐8 production, probably through inhibition of NF‐κB, and that they increase Akt phosphorylation in HepG2 cells.     

Evidence of cell-associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation

Abstract: Cell‐associated proteinases from Virgibacillus sp. SK33 isolated from fish sauce fermentation were extracted and characterized. Proteinases were effectively released when washed cells were incubated in 0.3 mg/mL lysozyme in 50 mM Tris‐maleate (pH 7) at 37 °C for 2 h. Major cell‐associated proteinases exhibited molecular mass of 17, 32, and 65 kDa, but only a 32‐kDa proteinase showed strong amidolytic activity toward Suc‐Ala‐Ala‐Pro‐Phe‐AMC. Activity of all cell‐associated proteinases was completely inhibited by phenylmethanesulfonyl fluoride, indicating a characteristic of serine proteinase. In addition, a 65‐kDa serine proteinase was also inhibited by ethylenediaminetetraacetic acid, implying a metal‐dependent characteristic. Optimum activity toward a synthetic peptide substrate was at 50 °C and pH 8 and 11. Proteinases with molecular mass of 17 and 32 kDa exhibited caseinolytic activity at 25% NaCl and activity based on a synthetic peptide substrate increased with NaCl concentrations up to 25%, suggesting their role in hydrolyzing proteins at high salt concentrations. This is the first report of liberated cell‐associated proteinases from a moderate halophile, Virgibacillus sp. Practical Application: The cell‐associated proteinases could be extracted from Virgibacillus sp. SK 33 using lysozyme. The extracted enzyme could be applied to hydrolyze food proteins at NaCl content as high as 25%. In addition, this study demonstrated that not only extracellular but also cell‐associated proteinases are key factors contributing to protein‐degrading ability at high salt environment of Virgibacillus sp. SK 33.     

Bilberry and its main constituents have neuroprotective effects against retinal neuronal damage in vitro and in vivo

Our aim was to determine whether a Vaccinium myrtillus (bilberry) anthocyanoside (VMA) and/or its main anthocyanidin constituents (cyanidin, delphinidin, and malvidin) can protect retinal ganglion cells (RGCs) against retinal damage in vitro and in vivo. In RGC cultures (RGC‐5, a rat ganglion cell‐line transformed using E1A virus) in vitro, cell damage and radical activation were induced by 3‐(4‐morpholinyl) sydnonimine hydrochloride (SIN‐1, a peroxynitrite donor). Cell viability was measured using a water‐soluble tetrazolium salt assay. Intracellular radical activation within RGC‐5 cells was evaluated using 5‐(and‐6)‐chloromethyl‐2,7‐dichlorodihydrofluorescein diacetate acetyl ester (CM‐H₂DCFDA). Lipid peroxidation was assessed using the supernatant fraction of mouse forebrain homogenates. In mice in vivo, we evaluated the effects of VMA on N‐methyl‐D ‐aspartic acid (NMDA)‐induced retinal damage using hematoxylin‐eosin and terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) stainings. VMA and all three anthocyanidins (i) significantly inhibited SIN‐1‐induced neurotoxicity and radical activation in RGC‐5, (ii) concentration‐dependently inhibited lipid peroxidation in mouse forebrain homogenates. Intravitreously injected VMA significantly inhibited the NMDA‐induced morphological retinal damage and increase in TUNEL‐positive cells in the ganglion cell layer. Thus, VMA and its anthocyanidins have neuroprotective effects (exerted at least in part via an anti‐oxidation mechanism) in these in vitro and in vivo models of retinal diseases.     

Isolation of a Recombinant Bacillus sp. TS‐23 α‐Amylase by Adsorption‐Elution on Raw Starch

A Bacillus sp. TS‐23 α‐amylase produced by recombinant Escherichia coli was adsorbed onto raw starch and the adsorbed enzyme was eluted with maltose or maltodextrin in 50 mM Tris/HCl buffer (pH 8.5). The adsorption‐elution procedure resulted in a yield of 53% α‐amylase activity and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS/PAGE) analysis showed that the eluted α‐amylase had a molecular mass of approximately 64 kDa. Raw starch could be used repeatedly in the adsorption‐ elution cycle with good reproducibility. Scanning electron microscopy of the isolated corn starch exhibited a smooth appearance of the granules before adsorption and only a small change in appearance after three adsorption‐elution cycles. These results suggest that the raw starch adsorption‐elution technique has a great potential in the isolation of Bacillus sp. TS‐23 α‐amylase from the culture broth of recombinant E. coli.     

Impact of Sod on the Expression of Stress‐Related Genes in Listeria monocytogenes 4b G with/without Paraquat Treatment

Listeria monocytogenes is a foodborne pathogen that causes listeriosis. Paraquat can generate reactive oxygen species (ROS) in cells, which results in oxidative stress. It was first shown that 1 mM of paraquat inhibited the growth rate of a superoxide dismutase (sod)‐deletion mutant (?sod) generated from L. monocytogenes 4b G but not in the wild‐type, and induced the expression of other resistance genes (kat, fri, perR, sigB, and recA) as well as sod in the wild type. Interestingly, without paraquat treatment the expression of all the 5 genes were repressed in ?sod compared to the wild type, while the expression of recA triggering SOS response, a global response to DNA damage, was increased in ?sod in the presence of 1 mM paraquat. Taken together, these results suggest that SOD plays a central role in oxidant defense of L. monocytogenes 4b G, and SOS probably significantly impacts ?sod survival under oxidative stress.     

New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

pHluorin is a pH‐sensitive variant of green fluorescent protein for measuring intracellular pH (pH_in) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pH_in for three prototrophic wild‐types. The new construct was also used to determine the pH_in in strains differing in the activity of the plasma membrane Pma1 H⁺‐ATPase and the influence of glucose on pH_in. We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands‐on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM /pH for wild‐type yeast and moderately decreased in mutants with affected potassium transport. Copyright © 2010 John Wiley & Sons, Ltd.     

Composition of cell walls isolated from cell types of grain sorghum stems

Cell types were isolated from sorghum stems at two stages of development, anthesis and grain maturity, to study cell wall characteristics. Cell walls were isolated from epidermis (EPID), sclerenchyma (SCL), vascular bundle zone (VBZ), inner vascular bundles (IVB) and pith parenchyma cells (PITH) and analysed for total carbohydrate, acid insoluble lignin, total uronosyls, neutral sugars and hydroxycinnamic acids. In addition, walls from SCL, VBZ, IVB and PITH were subjected to chemical fractionation to separate wall carbohydrate into polysaccharide groups. Although wall characteristics were similar at both plant maturities, there were differences in lignin concentration, hydroxycinnamic acids, and carbohydrate composition among the cell wall types. Lignin was lowest in the PITH walls (169 g kg⁻¹) and highest in SCL and EPID (c 211 g kg⁻¹). Cellulose was most abundant in VBZ and SCL walls with greater secondary wall formation. Pectic materials were most abundant in PITH walls. Xylans were similar among wall types except for EPID that contained higher amounts of xylose. Releasable hydroxycinnamates were not as consistent among the cell wall types. Total ferulates, including ester linked and releasable ether linked, tended to increase from PITH to SCL (8 to 15 g kg⁻¹ CW) with an increase in the proportion etherified within the wall matrices (PITH 51%; SCL 66%). Total p‐coumarates showed opposite trends with PITH walls having significantly more (35 g kg⁻¹ CW) than VBZ or SCL (19 and 13 g kg⁻¹ CW). EPID walls contained the least pCA (6.5 g kg⁻¹ CW). Except for the hydroxycinnamates, compositional trends for the different wall types would reflect changes from primary walls to increased amounts of secondary wall. Neutral sugar analysis of indigestible residues indicated similar carbohydrate compositions among the cell wall types, with xylose being less degradable than all other wall sugars. © 1999 Society of Chemical Industry